- Email: [email protected]
The timing of cell division: Ap4A as a signal
FRONTLINES
TIBS 23 – MAY 1998
The timing of cell division: Ap4A as a signal During the cell cycle of Escherichia coli, events such as DNA replication and cell division follow a strict timetable before two identical daughter cells are produced. The cell must therefore have a mechanism(s) for coordinating each event. For example, the interval between termination of DNA replication and cell division remains constant (20 min) at 37°C, under a variety of growth conditions1. If cells were to divide too early, the progeny would be smaller than normal and, worse still, replicating DNA strands could be torn apart and/or annucleoid cells could be produced. Cells lacking DNA, however, are remarkably rare in cultures of wild-type strains2 and the variation in cell size at the time of cell division is very small3. Most approaches to understanding the timing of cell division in a cell cycle have asked whether initiation of cell division and DNA replication are coupled4–10. In this article, I discuss evidence suggesting that Ap4A acts as a signal that couples these processes.
Isolation of cell-division mutants One approach to elucidating the timing of cell division in the normal cell cycle has been that of isolating mutants that can divide even when DNA replication is inhibited. DNA replication and cell division are coupled by two mechanisms: the first is the well-known SOS response, which is utilized when DNA replication is halted or DNA is damaged11, and which depends on the division inhibitor SfiA (also known as SulA) and its target SfiB (also known as SulB or FtsZ)12,13; the second is a putative pathway for delaying cell division until completion of DNA replication5–9. I found that in a temperature-sensitive, DNA-replication mutant, dnaB42, the SOS response is induced at a low level; it therefore seemed likely that mutants that can still divide when combined with the dnaB42 mutation encode altered versions of proteins normally involved in the cell cycle. As expected, this is the case for the novel mutants cfcA and cfcB, which are involved in replication–division coupling9: the cell number in cultures of the cfc dnaB42 double mutant continues to increase after a temperature shift to 41°C, but cell
division in the cfc1 dnaB42 strain (which lacks the cfc mutation) stops completely after 40 min at 41°C. Cell growth [measured by the increase in optical density at 660 nm (OD660)] is largely unaffected by the cfc mutations. However, while cfc1 dnaB42 cells consist of .10 mm filaments and contain one or two nucleoids, the cfc dnaB42 double mutant gives rise to short cells (3–5 mm filaments) with one or two nucleoids, and many small cells without DNA. Thus, the cfc mutations can induce cell division in the absence of DNA replication.
Early cell division At 30°C, under conditions that permit DNA replication, both the cell mass and the cell number of cfc1 dnaB42 and cfc dnaB42 double mutant cultures increase exponentially; their doubling times are also similar. Furthermore, the amount of protein in each culture is proportional to the OD660 and most cells are viable. However, the average number of cells per OD unit in the cfc dnaB42 double mutant cultures is about 40% higher than in the cfc1 dnaB42 cultures, and cfc dnaB42 double mutant cells are smaller. The cfc mutation therefore affects average cell size, but not the length of the cell cycle or the rate of increase in cell mass. Phase-fluorescence micrographs of DAPIstained cells show that, in culture, most cfc cells are at the single-nucleoid stage, but the mutants do not divide more than once per doubling of the nucleoid. Chromosome synthesis in wild-type strains begins when a cell reaches a certain fixed mass – the ‘initiation mass’14. The initiation mass, which is determined by directly observing the sizes both of the largest cells with a single nucleoid and of the smallest cells with a double nucleoid, is not altered by the cfc mutations. Furthermore, the cfc mutations affect neither the period of a round of DNA replication nor the DNA : mass ratio of growing cells. The most likely explanation for these results is that the cfc mutations cause early cell division, but affect neither DNA replication nor protein synthesis. The increase in cell number in the cfc mutants is due to shortening of the time interval
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00
between nucleoid division and cell division; however, there is a compensatory extension of the period from cell division to initiation of chromosome synthesis. The length of the period between the end of DNA replication and cell division in the cfc dnaB42 double mutant cells is calculated to be roughly 2 min, while that in the cfc1 dnaB4 cells is 20 min (see Fig. 1 and Ref. 15). Experiments using dnaB1 transductants show that the Cfc phenotype is not influenced by the dnaB42 mutation.
Ap4A levels The cfcA mutant has a missense mutation in the glySa gene, which encodes the a subunit of glycyl-tRNA synthetase; cfcB mutants have an IS2 insertion in the apaH coding region15. The apaH gene encodes Ap4A hydrolase16, and the Ap4A concentration could therefore be increased by the mutation. A plasmid containing the (wild-type) apaH gene complements cfcA, suggesting that the protein product of the gene defective in cfcA is involved in the synthesis of Ap4A. Twodimensional, thin-layer chromatography of 32P-labeled compounds in formic-acidsoluble fractions of the cfc mutants and their parent strain shows that cfcA cells produce about 15-fold more Ap4A than the parent strain, and that cfcB cells produce about 100-fold more Ap4A than the parent strain (Fig. 2). Ap4A cannot be detected in cfcA or cfcB mutant strains that have been transformed with the (wildtype) apaH gene15. High levels of Ap4A could therefore be a signal that causes early cell division in cfc mutants. While the effect of apaH on the level of Ap4A is straightforward, the effects of glyS are complex. In vitro, glycyl adenylate intermediates that are bound to glycyltRNA synthetase donate AMP to ATP, yielding Ap4A17. Glycyl-tRNA synthetase also catalyses the PPi-dependent conversion of Ap4A to ADP18. Thus, the increase in Ap4A concentration observed in the cfcA mutant might be caused either by enhanced synthesis of Ap4A, or by inhibition of the conversion of Ap4A to ADP. The existence of such reactions in vivo, however, has not been demonstrated. Further studies are therefore required in order to elucidate how glyS affects the cellular levels of Ap4A.
Signals in normal division When the cfcB mutation is introduced into wild-type E. coli (as opposed to the dnaB42 strain), transductants display the Cfc phenotype: small cells are produced when transductants are cultivated in a liquid medium for several generations.
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FRONTLINES (a)
(b)
Normal (cfc +)
Normal (cfc +) 2
2
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Initiation of DNA replication
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TIBS 23 – MAY 1998
Figure 1 Comparisons of the cell cycles of cfc mutants and their parent strain. (a) Schematic representations of these cell cycles, showing premature division of the cfc cells and illustrating the compensating increase in the interval between cell division and the initiation of DNA replication. (b) Theoretical change in cell number during the division cycle. The black lines show cell number and the blue lines show OD660 as a function of time, during the division cycle. C is the period in which a round of DNA replication takes place; D is the period from the end of DNA replication to cell division; B is the period from the birth of a cell to the initiation of DNA replication. In cells lacking the cfc mutation (cfc1), C and D are constant (40 min and 20 min, respectively) and the length of the cell cycle is 65 min; B is therefore 5 min. In the cfc mutants, the length of the cell cycle is ~70 min, and C is unaffected by the cfc mutations. Therefore, D 1 B should be 30 min. To cause a 40% increase in cell number, the integral of the cfc strain cell number should be 40% higher (see shadowed region) than before. B and D can therefore be calculated to be 28 min and 2 min, respectively. Figure reproduced, with permission, from Ref. 15.
Figure 2 Two-dimensional thin-layer chromatography of cellular nucleotides of cfc mutants and the parent strain visualized by UV260 absorbance. For experimental details see Ref. 15. Arrows indicate Ap4A spots. Figure reproduced, with permission, from Ref. 15.
After overnight cultivation, however, the phenotype is lost. Transformation of wildtype cells with the (wild-type) apaH gene causes a reduction in the frequency of cell division for the first several generations
158
in a liquid medium and produces longer cells, but again the phenotype is lost after overnight cultivation. By contrast, the cfcB transductants of the parental (dnaB42) strain exhibit the Cfc phenotype
continuously. In conclusion, an increase in the cellular concentration of Ap4A (due to the cfcB mutation, for example) causes early cell division, while a decrease (due to overexpression of wild-type apaH) causes delayed cell division; however, maintenance of these phenotypes depends on the genetic background of the parental cell15. Ap4A was first discovered in an in vitro reaction system17 and was subsequently found in vivo in prokaryotic and eukaryotic cells19,20. It might play a role as an ‘alarmone’ in bacteria, because its concentration increases in response to a variety of stresses19,21, such as heat shock or oxidative stress. Its role in cellular adaptation to stress has, however, been questioned22. Ap4A has also been proposed to be a signal that links protein synthesis to initiation of DNA replication. This idea stems from two observations: cell-cycle-dependent fluctuation in intracellular levels of Ap4A in eukaryotes19 and induction of multiple replication eyes following addition of Ap4A to permeabilized resting cells23. Contradictory results suggest that Ap4A is associated with cell-contact-dependent growth inhibition24. More recent work suggests that the Ap3A : Ap4A ratio has opposing effects on cell differentiation and apoptosis in human cell cultures25. Clearly, although Ap4A is a nucleotide present in a variety of cells, its biological role remains obscure. Early work in E. coli showed that, in an apaH apaG ksgA triple mutant, a kasugamycin-induced block on protein synthesis and cell division could be relieved by overexpression of (wild-type) ApaH26. The authors suggested that Ap4A interacts with the ribosome machinery, inhibiting protein synthesis and cell division (during cellular adaptation in response to stress). This observation would appear to contradict our finding that Ap4A induces cell division. However, the effect of Ap4A on cell division seen in these early studies is more likely to be caused by an indirect and nonspecific effect resulting from inhibition of protein synthesis – as opposed to any specific connection between Ap4A and cell division. In our work, the elevation of Ap4A concentration observed in the cfc mutants induces cell division, but does not affect the period of a round of DNA replication, the DNA : mass ratio of growing cells, or the initiation mass for DNA replication15. Interestingly, Ap4A levels (measured every 15 min) remain constant throughout the cell cycle in synchronized cells27; however, it is possible that
FRONTLINES
TIBS 23 – MAY 1998 a transient flux of Ap4A would not be detected in such experiments. One possible explanation for how Ap4A affects cell division is that it binds to a protein essential for cell division and alters its function. The loss of phenotype after overnight cultivation, in the above experiments, might be explained by a suppressor mutation or physiological change that affects the binding of Ap4A to such a target. Introduction of the cfcA mutation into wild-type E. coli yields cells that display the Cfc phenotype. This phenotype is not lost – even after three cycles of overnight cultivation. The presence of a multicopy plasmid carrying a glySa gene that possesses the cfcA mutation also yields a permanent Cfc phenotype. By contrast, the presence of a multicopy plasmid carrying the (wildtype) glySa gene inhibits cell division of wild-type cells and produces filamentous cells under normal growth conditions15. I therefore speculate that GlySa plays an important role in the normal cell cycle and cooperates with Ap4A.
Conclusions E. coli cells possessing cfc mutations divide before they reach the size at which cfc1 cells divide, and produce many small cells – each with a single nucleoid. The mutations affect the timing of cell division, but not the initiation mass for chromosome replication. This is because
of a reduction in the length of the period between nuclear division and cell division, and a compensatory increase in the time interval between cell division and initiation of the next round of DNA replication. These mutations relieve cells from division arrest caused by the inhibition of DNA replication. As such, the cfc mutants are defective in a signaling mechanism that couples DNA replication and cell division. I conclude that Ap4A is part of this signaling mechanism.
Acknowledgements I am grateful to Jun-ichi Tomizawa for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, from the Ministry of Education, Science, Sports and Culture of Japan.
References 1 Helmstetter, C. E. and Cooper, S. (1968) J. Mol. Biol. 31, 507–518 2 Howe, W. E. and Mount, D. W. (1975) J. Bacteriol. 124, 1113–1121 3 Schaechter, M., Williamson, J. P., Hood, J. R. J. and Koch, A. L. (1962) J. Gen. Microbiol. 29, 421–434 4 Inouye, M. (1971) J. Bacteriol. 106, 539–542 5 Jones, N. C. and Donachie, W. D. (1973) Nat. New Biol. 243, 100–103 6 Burton, P. and Holland, I. B. (1983) Mol. Gen. Genet. 190, 309–314 7 Tormo, A., Cabrera, C. F. and Vicente, M. (1985) J. Gen. Chem. 258, 10637–10641 8 Jaffé, A., D’Ari, R. and Norris, V. (1986) J. Bacteriol. 165, 66–71
Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts The mechanisms of physiological cell death (apoptosis) are conserved across metazoan phyla. For example, the mammalian cell-death inhibitory gene bcl-2 has been shown to prevent programmed cell death when expressed in Caenorhabditis elegans, and proteins encoded by the C. elegans genes ced-9 and ced-3 have been shown to resemble the mammalian cell-death proteins Bcl-2 and apoptotic cysteine proteases (caspases), structurally and functionally1–3. While there
have been reports of physiological cell death in single-celled organisms, until now no homologs of metazoan cell-death genes have been found in unicellular organisms. Here, we describe the identification of new members of the inhibitor of apoptosis (IAP) family of proteins in a number of organisms, including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. IAP proteins are a class of cell-death inhibitors that function in insects and
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00
9 Nishimura, A. (1989) Mol. Gen. Genet. 215, 286–293 10 Bernander, R. and Nordström, K. (1990) Cell 60, 365–374 11 Little, J. and Mount, D. W. (1982) Cell 29, 11–22 12 Huisman, O. and D’Ari, R. (1981) Nature 290, 797–799 13 George, J., Castellazi, M. and Buttin, G. (1975) Mol. Gen. Genet. 140, 309–332 14 Donachie, W. D. (1968) Nature 219, 1077–1079 15 Nishimura, A. et al. (1997) Genes Cells 2, 401–413 16 Mechulam, Y. et al. (1985) J. Bacteriol. 164, 63–69 17 Zamecnik, P. C., Stephenson, M. L., Janeway, C. M. and Randerath, K. (1966) Biochem. Biophys. Res. Commun. 24, 98–105 18 Led, J. J., Switon, W. K. and Jensen, K. F. (1983) Eur. J. Biochem. 136, 469–479 19 Zamecnik, P. (1983) Anal. Biochem. 134, 1–10 20 Bochner, B. R. et al. (1984) Cell 37, 225–232 21 Lee, P. C., Bochner, B. N. and Ames, B. N. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7496–7500 22 Plateau, P., Fromant, M. and Blanquet, S. (1987) J. Bacteriol. 169, 3817–3820 23 Grummt, F. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 371–375 24 Segal, E. and Le Pecq, J. B. (1986) Exp. Cell Res. 167, 119–126 25 Vartanian, A. et al. (1997) FEBS Lett. 415, 160–162 26 Lévêque, F. et al. (1989) J. Mol. Biol. 212, 319–329 27 Plateau, P., Fromant, M., Kepes, F. and Blanquet, S. (1987) J. Bacteriol. 169, 419–422
AKIKO NISHIMURA National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan. Email: [email protected]
vertebrates to inhibit apoptosis during development, as well as that induced by viral infection, drugs, tumor necrosis factor (TNF) and over-expression of caspases4–12. The first IAP genes isolated were the baculovirus genes CpIAP (from Cydia pomonella granulosis virus, CpGV) and OpIAP (from Orgyia pseudotsugata nuclear polyhedrosis virus, OpNPV). They were found by virtue of their ability to compensate for loss of function of the caspase inhibitor protein p35 in Autographa Californica nuclear polyhedrosis virus (AcNPV) mutants4,5. It is believed that IAPs are used by baculoviruses to prevent a defensive apoptotic response of the host cells that would otherwise limit viral replication13. More recently, numerous cellular homologs of IAPs have been described. The Drosophila genome encodes two IAP genes Diap1 and Diap2. Transgenic expression of these genes was able to suppress developmental death as well as death caused by overexpression of the reaper or head involution defective genes7. Mammalian
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TIBS 23 – MAY 1998
The timing of cell division: Ap4A as a signal During the cell cycle of Escherichia coli, events such as DNA replication and cell division follow a strict timetable before two identical daughter cells are produced. The cell must therefore have a mechanism(s) for coordinating each event. For example, the interval between termination of DNA replication and cell division remains constant (20 min) at 37°C, under a variety of growth conditions1. If cells were to divide too early, the progeny would be smaller than normal and, worse still, replicating DNA strands could be torn apart and/or annucleoid cells could be produced. Cells lacking DNA, however, are remarkably rare in cultures of wild-type strains2 and the variation in cell size at the time of cell division is very small3. Most approaches to understanding the timing of cell division in a cell cycle have asked whether initiation of cell division and DNA replication are coupled4–10. In this article, I discuss evidence suggesting that Ap4A acts as a signal that couples these processes.
Isolation of cell-division mutants One approach to elucidating the timing of cell division in the normal cell cycle has been that of isolating mutants that can divide even when DNA replication is inhibited. DNA replication and cell division are coupled by two mechanisms: the first is the well-known SOS response, which is utilized when DNA replication is halted or DNA is damaged11, and which depends on the division inhibitor SfiA (also known as SulA) and its target SfiB (also known as SulB or FtsZ)12,13; the second is a putative pathway for delaying cell division until completion of DNA replication5–9. I found that in a temperature-sensitive, DNA-replication mutant, dnaB42, the SOS response is induced at a low level; it therefore seemed likely that mutants that can still divide when combined with the dnaB42 mutation encode altered versions of proteins normally involved in the cell cycle. As expected, this is the case for the novel mutants cfcA and cfcB, which are involved in replication–division coupling9: the cell number in cultures of the cfc dnaB42 double mutant continues to increase after a temperature shift to 41°C, but cell
division in the cfc1 dnaB42 strain (which lacks the cfc mutation) stops completely after 40 min at 41°C. Cell growth [measured by the increase in optical density at 660 nm (OD660)] is largely unaffected by the cfc mutations. However, while cfc1 dnaB42 cells consist of .10 mm filaments and contain one or two nucleoids, the cfc dnaB42 double mutant gives rise to short cells (3–5 mm filaments) with one or two nucleoids, and many small cells without DNA. Thus, the cfc mutations can induce cell division in the absence of DNA replication.
Early cell division At 30°C, under conditions that permit DNA replication, both the cell mass and the cell number of cfc1 dnaB42 and cfc dnaB42 double mutant cultures increase exponentially; their doubling times are also similar. Furthermore, the amount of protein in each culture is proportional to the OD660 and most cells are viable. However, the average number of cells per OD unit in the cfc dnaB42 double mutant cultures is about 40% higher than in the cfc1 dnaB42 cultures, and cfc dnaB42 double mutant cells are smaller. The cfc mutation therefore affects average cell size, but not the length of the cell cycle or the rate of increase in cell mass. Phase-fluorescence micrographs of DAPIstained cells show that, in culture, most cfc cells are at the single-nucleoid stage, but the mutants do not divide more than once per doubling of the nucleoid. Chromosome synthesis in wild-type strains begins when a cell reaches a certain fixed mass – the ‘initiation mass’14. The initiation mass, which is determined by directly observing the sizes both of the largest cells with a single nucleoid and of the smallest cells with a double nucleoid, is not altered by the cfc mutations. Furthermore, the cfc mutations affect neither the period of a round of DNA replication nor the DNA : mass ratio of growing cells. The most likely explanation for these results is that the cfc mutations cause early cell division, but affect neither DNA replication nor protein synthesis. The increase in cell number in the cfc mutants is due to shortening of the time interval
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00
between nucleoid division and cell division; however, there is a compensatory extension of the period from cell division to initiation of chromosome synthesis. The length of the period between the end of DNA replication and cell division in the cfc dnaB42 double mutant cells is calculated to be roughly 2 min, while that in the cfc1 dnaB4 cells is 20 min (see Fig. 1 and Ref. 15). Experiments using dnaB1 transductants show that the Cfc phenotype is not influenced by the dnaB42 mutation.
Ap4A levels The cfcA mutant has a missense mutation in the glySa gene, which encodes the a subunit of glycyl-tRNA synthetase; cfcB mutants have an IS2 insertion in the apaH coding region15. The apaH gene encodes Ap4A hydrolase16, and the Ap4A concentration could therefore be increased by the mutation. A plasmid containing the (wild-type) apaH gene complements cfcA, suggesting that the protein product of the gene defective in cfcA is involved in the synthesis of Ap4A. Twodimensional, thin-layer chromatography of 32P-labeled compounds in formic-acidsoluble fractions of the cfc mutants and their parent strain shows that cfcA cells produce about 15-fold more Ap4A than the parent strain, and that cfcB cells produce about 100-fold more Ap4A than the parent strain (Fig. 2). Ap4A cannot be detected in cfcA or cfcB mutant strains that have been transformed with the (wildtype) apaH gene15. High levels of Ap4A could therefore be a signal that causes early cell division in cfc mutants. While the effect of apaH on the level of Ap4A is straightforward, the effects of glyS are complex. In vitro, glycyl adenylate intermediates that are bound to glycyltRNA synthetase donate AMP to ATP, yielding Ap4A17. Glycyl-tRNA synthetase also catalyses the PPi-dependent conversion of Ap4A to ADP18. Thus, the increase in Ap4A concentration observed in the cfcA mutant might be caused either by enhanced synthesis of Ap4A, or by inhibition of the conversion of Ap4A to ADP. The existence of such reactions in vivo, however, has not been demonstrated. Further studies are therefore required in order to elucidate how glyS affects the cellular levels of Ap4A.
Signals in normal division When the cfcB mutation is introduced into wild-type E. coli (as opposed to the dnaB42 strain), transductants display the Cfc phenotype: small cells are produced when transductants are cultivated in a liquid medium for several generations.
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(b)
Normal (cfc +)
Normal (cfc +) 2
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Cell number
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D 40 42 Time (min)
B 70
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Initiation of DNA replication
Cell number
Cell division
Cell mass (OD660, log scale)
TIBS 23 – MAY 1998
Figure 1 Comparisons of the cell cycles of cfc mutants and their parent strain. (a) Schematic representations of these cell cycles, showing premature division of the cfc cells and illustrating the compensating increase in the interval between cell division and the initiation of DNA replication. (b) Theoretical change in cell number during the division cycle. The black lines show cell number and the blue lines show OD660 as a function of time, during the division cycle. C is the period in which a round of DNA replication takes place; D is the period from the end of DNA replication to cell division; B is the period from the birth of a cell to the initiation of DNA replication. In cells lacking the cfc mutation (cfc1), C and D are constant (40 min and 20 min, respectively) and the length of the cell cycle is 65 min; B is therefore 5 min. In the cfc mutants, the length of the cell cycle is ~70 min, and C is unaffected by the cfc mutations. Therefore, D 1 B should be 30 min. To cause a 40% increase in cell number, the integral of the cfc strain cell number should be 40% higher (see shadowed region) than before. B and D can therefore be calculated to be 28 min and 2 min, respectively. Figure reproduced, with permission, from Ref. 15.
Figure 2 Two-dimensional thin-layer chromatography of cellular nucleotides of cfc mutants and the parent strain visualized by UV260 absorbance. For experimental details see Ref. 15. Arrows indicate Ap4A spots. Figure reproduced, with permission, from Ref. 15.
After overnight cultivation, however, the phenotype is lost. Transformation of wildtype cells with the (wild-type) apaH gene causes a reduction in the frequency of cell division for the first several generations
158
in a liquid medium and produces longer cells, but again the phenotype is lost after overnight cultivation. By contrast, the cfcB transductants of the parental (dnaB42) strain exhibit the Cfc phenotype
continuously. In conclusion, an increase in the cellular concentration of Ap4A (due to the cfcB mutation, for example) causes early cell division, while a decrease (due to overexpression of wild-type apaH) causes delayed cell division; however, maintenance of these phenotypes depends on the genetic background of the parental cell15. Ap4A was first discovered in an in vitro reaction system17 and was subsequently found in vivo in prokaryotic and eukaryotic cells19,20. It might play a role as an ‘alarmone’ in bacteria, because its concentration increases in response to a variety of stresses19,21, such as heat shock or oxidative stress. Its role in cellular adaptation to stress has, however, been questioned22. Ap4A has also been proposed to be a signal that links protein synthesis to initiation of DNA replication. This idea stems from two observations: cell-cycle-dependent fluctuation in intracellular levels of Ap4A in eukaryotes19 and induction of multiple replication eyes following addition of Ap4A to permeabilized resting cells23. Contradictory results suggest that Ap4A is associated with cell-contact-dependent growth inhibition24. More recent work suggests that the Ap3A : Ap4A ratio has opposing effects on cell differentiation and apoptosis in human cell cultures25. Clearly, although Ap4A is a nucleotide present in a variety of cells, its biological role remains obscure. Early work in E. coli showed that, in an apaH apaG ksgA triple mutant, a kasugamycin-induced block on protein synthesis and cell division could be relieved by overexpression of (wild-type) ApaH26. The authors suggested that Ap4A interacts with the ribosome machinery, inhibiting protein synthesis and cell division (during cellular adaptation in response to stress). This observation would appear to contradict our finding that Ap4A induces cell division. However, the effect of Ap4A on cell division seen in these early studies is more likely to be caused by an indirect and nonspecific effect resulting from inhibition of protein synthesis – as opposed to any specific connection between Ap4A and cell division. In our work, the elevation of Ap4A concentration observed in the cfc mutants induces cell division, but does not affect the period of a round of DNA replication, the DNA : mass ratio of growing cells, or the initiation mass for DNA replication15. Interestingly, Ap4A levels (measured every 15 min) remain constant throughout the cell cycle in synchronized cells27; however, it is possible that
FRONTLINES
TIBS 23 – MAY 1998 a transient flux of Ap4A would not be detected in such experiments. One possible explanation for how Ap4A affects cell division is that it binds to a protein essential for cell division and alters its function. The loss of phenotype after overnight cultivation, in the above experiments, might be explained by a suppressor mutation or physiological change that affects the binding of Ap4A to such a target. Introduction of the cfcA mutation into wild-type E. coli yields cells that display the Cfc phenotype. This phenotype is not lost – even after three cycles of overnight cultivation. The presence of a multicopy plasmid carrying a glySa gene that possesses the cfcA mutation also yields a permanent Cfc phenotype. By contrast, the presence of a multicopy plasmid carrying the (wildtype) glySa gene inhibits cell division of wild-type cells and produces filamentous cells under normal growth conditions15. I therefore speculate that GlySa plays an important role in the normal cell cycle and cooperates with Ap4A.
Conclusions E. coli cells possessing cfc mutations divide before they reach the size at which cfc1 cells divide, and produce many small cells – each with a single nucleoid. The mutations affect the timing of cell division, but not the initiation mass for chromosome replication. This is because
of a reduction in the length of the period between nuclear division and cell division, and a compensatory increase in the time interval between cell division and initiation of the next round of DNA replication. These mutations relieve cells from division arrest caused by the inhibition of DNA replication. As such, the cfc mutants are defective in a signaling mechanism that couples DNA replication and cell division. I conclude that Ap4A is part of this signaling mechanism.
Acknowledgements I am grateful to Jun-ichi Tomizawa for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, from the Ministry of Education, Science, Sports and Culture of Japan.
References 1 Helmstetter, C. E. and Cooper, S. (1968) J. Mol. Biol. 31, 507–518 2 Howe, W. E. and Mount, D. W. (1975) J. Bacteriol. 124, 1113–1121 3 Schaechter, M., Williamson, J. P., Hood, J. R. J. and Koch, A. L. (1962) J. Gen. Microbiol. 29, 421–434 4 Inouye, M. (1971) J. Bacteriol. 106, 539–542 5 Jones, N. C. and Donachie, W. D. (1973) Nat. New Biol. 243, 100–103 6 Burton, P. and Holland, I. B. (1983) Mol. Gen. Genet. 190, 309–314 7 Tormo, A., Cabrera, C. F. and Vicente, M. (1985) J. Gen. Chem. 258, 10637–10641 8 Jaffé, A., D’Ari, R. and Norris, V. (1986) J. Bacteriol. 165, 66–71
Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts The mechanisms of physiological cell death (apoptosis) are conserved across metazoan phyla. For example, the mammalian cell-death inhibitory gene bcl-2 has been shown to prevent programmed cell death when expressed in Caenorhabditis elegans, and proteins encoded by the C. elegans genes ced-9 and ced-3 have been shown to resemble the mammalian cell-death proteins Bcl-2 and apoptotic cysteine proteases (caspases), structurally and functionally1–3. While there
have been reports of physiological cell death in single-celled organisms, until now no homologs of metazoan cell-death genes have been found in unicellular organisms. Here, we describe the identification of new members of the inhibitor of apoptosis (IAP) family of proteins in a number of organisms, including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. IAP proteins are a class of cell-death inhibitors that function in insects and
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00
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AKIKO NISHIMURA National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan. Email: [email protected]
vertebrates to inhibit apoptosis during development, as well as that induced by viral infection, drugs, tumor necrosis factor (TNF) and over-expression of caspases4–12. The first IAP genes isolated were the baculovirus genes CpIAP (from Cydia pomonella granulosis virus, CpGV) and OpIAP (from Orgyia pseudotsugata nuclear polyhedrosis virus, OpNPV). They were found by virtue of their ability to compensate for loss of function of the caspase inhibitor protein p35 in Autographa Californica nuclear polyhedrosis virus (AcNPV) mutants4,5. It is believed that IAPs are used by baculoviruses to prevent a defensive apoptotic response of the host cells that would otherwise limit viral replication13. More recently, numerous cellular homologs of IAPs have been described. The Drosophila genome encodes two IAP genes Diap1 and Diap2. Transgenic expression of these genes was able to suppress developmental death as well as death caused by overexpression of the reaper or head involution defective genes7. Mammalian
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