Cur~Gen~s
Current Genetics2, 229-232 (1980)
© bySpringer-Verlag 1980
Short Communication Net Synthesis of Chloroplast DNA Throughout the Synchronized Vegetative Cell-Cycle of Chlamydomonas Monique Turmel, Claude Lemieux, and Robert W. Lee Department of Biology,DalhousieUniversity,Halifax,Nova Scotia B3If4JI Canada
Summary. The accumulation of chloroplast and nuclear DNAs during the 12 h light and 12 h dark synchronized vegetative cell-cycle of Chlamydomonas reinhardtii was monitored by the direct optical quantification of these DNAs in the analytical ultracentrifuge. Net synthesis of nuclear DNA was sharply discontinuous and this synthesis occurred during the first 6 h of the dark period. In contrast, the net synthesis of chloroplast DNA appeared continuous throughout the cell-cycle. The rate of this accumulation, however, was greatest in the dark period.
Key words: Chlamydomonas - chloroplast DNA -cellcycle
Introduction Two major DNA components widely differing in base composition are recovered from the unicellular alga C. reinhardtii. One of these, termed a, with a density of 1.723 g/mi, represents most of the cellular DNA and is of nuclear origin (Sueoka et al. 1967; Robreau and Le Gal 1975). The second major DNA component, termed/3, with a density of 1.696 g/ml, is associated with the single chloroplast of this alga (Sager and Ishida 1963; Dron et al. 1979). It usually represents about 10% of the total cellular DNA (Sueoka et al. 1967). This DNA, hereafter called chloroplast DNA (cpDNA), has a molecular weight of 1.34 x 108 daltons (cf. Behn and Herrmann 1977) and the cellular content of cpDNA indicates that there are many copies per haploid vegetative cell. The time course of many cellular events associated with the synchronized vegetative growth of C. reinhardtii Offprint requests to: R. W. Lee
under alternating 12 h light and 12 h dark periods has been well documented (Kates et al. 1968; Mihara and Hase 1971; Howell 1972; Howell and Walker 1977). Cell growth (increment in size) occurs during the light period, while nuclear DNA synthesis, mitosis, and cell division occur in the dark period. The release of daughter cells from the parental cell wall occurs near the end of the dark period or the first hour of the light period. The number of daughter cells produced from a single parent cell (typically 4, 8, or more) is primarily a function of light intensity and hence is influenced by culture density (cf. Lien and Knutsen 1979). Chiang and Sueoka (1967), using l S N J 4 N density transfer techniques similar to those of Meselson and Stahl (1958), examined the pattern and timing of cpDNA replication during the light: dark synchronized vegetative growth of C. reinhardtii. In a cell-cycle during which cell number increased four-fold, most or all cpDNA replication was restricted to the light period as revealed by at least two semiconservative duplications of all cpDNA molecules, while two semiconservative duplications of all nuclear DNA molecules occurred specifically in the dark period. In similar density transfer experiments with synchronized cultures of C. reinhardtii, Lee and Jones (1973) were unable to confirm the classical semiconservative replication pattern for cpDNA as described by Chlang and Sueoka (1967). Rather, after transfer to 14N_ medium only one unimodal cpDNA band was ever visible and this shifted gradually towards lighter densities with continued growth in 14N-medium. Similar replication patterns after density transfer have been reported for the mitochondrial (mt) DNAs of yeast (Williamson and Fennell 1974) and Euglena (Richards and Ryan 1974). More importantly in terms of this communication, the data of Lee and Jones (1973) reveal less synchrony in the cell-cycle timing ofcpDNA replication than described by Chiang and Sueoka (1967). Although the most O172-8083/80/0002/0229/$01.00
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of C. reinhardtii by direct optical quantification of this DNA in the analytical ultracentrifuge. The results, free of ambiguities frequently associated with isotopic labelling experiments, indicate that net cpDNA synthesis is continuous across the cell-cycle.
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Time (hr) Fig. 1. Increase of chloroplast and nuclear DNAs during the synchronized growth of C. reinhardtii (137c, mt+). An 81 culture was synchronized by growth under alternating 12 h light and 12 h dark periods and DNA was extracted and quantified directly in the analytical ultracentrifuge relative to Bacillus subtilis phage SP8 DNA (p = 1.742 g/ml) which was added to the lysates prior to DNA extraction. Details of culture, DNA extraction, and DNA quantification have been described previously (Lemieux et al. 1980). Samples for DNA extraction were taken from volumes of culture containing 2.0-5.0 x 108 cells except that volumes taken from hours-18 and -21 were 2- to 4-times smaller than the volumes taken previously at hour-!2. With the onset of light in cycle-6, the culture density had reached 2.8 x 105 cells/ml. The strain of C. reinhardtii employed here was obtained from Drs. N. W. Gillham and J. E. Boynton who in turn obtained it from Dr. R. P. Levine and hence it appears to be the same one used in the density transfer experiment of Chiang and Sueoka (1967)
dramatic density shifts in cpDNA were detected in the light period, further important shifts were also detected during the dark period but the quantitative significance of these shifts was difficult to access as the cpDNA density approached that of fully 14N-labelled molecules. More recently, Chiang and coworkers (Chiang 1975; Grant et al. 1978) using radioactive DNA precursors have found major labelling of C. reinhardtii cpDNA during both the light and dark phases of synchronized vegetative culture. In an attempt to relate these results to their earlier report (Chiang and Sueoka 1967), it was suggested that the light period incorporation results from net cpDNA synthesis and that repair o f cpDNA may account for the dark period incorporation. In this study we have followed the accumulation of cpDNA during the synchronized vegetative cell-cycle
Results and Discussion Data on the accumulation of cp and nuclear DNAs during the light: dark synchronized growth of C reinhardtii are shown in Fig 1. After five sucessive light: dark cycles, samples were harvested for DNA extraction at various times commencing with the dark period of cycle-6 and ending with the light period of cycle-8, as shown. In cycle-6 and -7, cell number increased 6.9and 3.6-fold during the respective dark periods while nuclear DNA levels, monitored more frequently, showed comparable increases during the first half of these dark periods. In contrast, the accumulation of cpDNA during both the light and dark periods of culture is evident in the samples taken from the dark period of cycle-6 through the dark period of cycle-7. The rate of cpDNA accumulation, however, in both this and other experiments, consistently appears most intense in the dark period during an 8 - 1 0 h interval which lags behind but overlaps with the interval of nuclear DNA synthesis. The period of most intense cpDNA accumulation occurs during the interval of the light: dark cycle when chloroplast division and cytokinesis reportedly take place (Mihara and Hase 1971). The negligible accumulation of cpDNA during the light period of cycle-8 probably resulted from the culture nearing stationary phase. Most cells failed to divide in the dark period which followed. Table 1 reports the average cellular content of total DNA and cpDNA from the culture times of Fig. 1 where cell counts were determined. The percentage of total DNA represented by the cpDNA fraction is reported for all culture times. These data reveal significant cellcycle fluctuations in the percentage of cpDNA as expected from the different cell-cycle synthesis patterns of cp and nuclear DNA, as shown in Fig. 1. Among the cell-cycle times examined, the percentage of cpDNA is maximum (16%-18%) at the end of the light period (hour-12) just before nuclear DNA synthesis and minimum (5%-9%) after three hours of darkness (hour-18) when nuclear DNA synthesis has reduced its proportion. Once again, the hour-12 value of cycle-8 is not representative as the culture was nearing stationary phase. The average cpDNA content at 2 0 - 2 2 x 10 -15 g/cell among cells harvested at the onset of the light period (hour.24/0) is in agreement with previous reports (Lemieux et al. 1980; Whiteway and Lee 1977) and indicates an average cpDNA copy number of about 100 copies per cell. These
M. Turmel et al.: Chloroplast DNA Synthesis
231
Table 1. Cell-cycle changes in total DNA (nuclear DNA plus cpDNA) and cpDNA during the synchronized vegetative growth of C. reinhardtii (from Fig. 1). Determinations of DNA content per eell are corrected for minor cell loses during the cell harvesting and washing steps and epDNA copy number per cell is based on the molecular weight of individual molecules being 134 megadaltons (Behn and Herrmann 1977) Cycle
Timea (h)
Total e p D N A / epDNA DNA/cell cell copies/ (gx1015 ) (gx1015 ) cell
% cpDNA
6 6 6/7 7 7 7 7 7 7/8
12 18 24/0 6 12 15 18 21 24/0
246 185 209 197
8
3
-
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8
12
203
25.1
113
17.5 5.3 10.9 16.3 16.7 11.0 8.6 10.8 11.1 12.8 12.4
43.0 20.2 34.9 22.1
194 91 157 100
Hour 0 = onset of the light period; 6 = midpoint of the light period; 12 - light: dark transition point; 18 = midpoint of the dark period; 24/0 = dark: light transition point (i.e. beginning of a new cycle)
values are undoubtedly lower in the last half of the dark period just after cell division and they obviously increase throughout the light period, reaching maximum in the first half of the dark period just before cell division. Similar experiments with synchronized Chlamydomonas moewusii also reveal net synthesis of cpDNA during both the light and dark phases of culture (unpublished observations) and the cpDNA copy number per cell at, the dark:light transition of culture is also about 10.0 (Lemieux et al. 1980). The data we present on the timing of DNA synthesis during the synchronized vegetative culture of C. reinhardtii are the first in which the changing levels of cp and nuclear DNAs, expressed per ml of culture, were monitored directly by the UV absorption of these DNAs in the analytical ultracentrifuge. Evidence for the efficient extraction of these DNAs which together account for over 96% of the cellular DNA of C reinhardtii (Sueoka et al. 1967) is provided by the correspondence between the combined cp and nuclear DNA levels per cell which we recovered (Table 1), and the range of values reported for whole cell DNA as determined by colorimetric assays with similarly grown cultures (Sueoka e t al. 1967; Whiteway and Lee 1977). Moreover, the temporal pattern of nuclear DNA synthesis as revealed by the approach used in this study is indistinguishable from that obtained by independent methods with similar cultures of this alga (Kates et al. 1968).
The continuous net synthesis of cpDNA which we describe makes it unlikely that each cellular doubling of cpDNA results from a single replication of each individual cpDNA molecule. Assuming that the synthesis of an individual cpDNA molecule occupies only a fraction of the cell-cycle, it is hard to envisage a mechanism by which cells could distinguish those molecules which had already been replicated from those which had not. We, therefore, favor the possibility that the cpDNA molecules of mitotically dividing C. reinhardtii are randomly selected for replication as indicated in studies with the mtDNA of cultured mouse L cells (Bogenhagen and Clayton 1977; see also, Birky 1978). Our inability to discriminate newly replicated from unreplicated cpDNA molecules in density transfer experiments, as mentioned above, makes it extremely difficult to test this hypothesis directly. It is noteworthy that the interval of maximal cpDNA accumulation in C. reinhardtii was detected after the onset of nuclear DNA replication had reduced its proportion to a cell-cycle minimum and that minimal cpDNA accumulation was observed prior to nuclear DNA replication when the proportion of cpDNA had reached a cell-cycle maximum. On the basis of these data, it is tempting to suggest that the rate of cpDNA synthesis occurs in response to a changing cpDNA/nuclear DNA ratio. Such an hypothesis is consistent with the observation that cpDNA content increases with nuclear ploidy in haploid and diploid vegetative cell cultures of C. reinhardtii (Whiteway and Lee 1977). Similar results have been reported for the mtDNA of yeast (Grimes et al. 1974). Alternatively, cell volume, which also increases with nuclear ploidy in both organisms, might be the factor most crucial to the regulation of organelle DNA synthesis (cf. Williamson et al. 1977; Lee and Johnson 1977). Evidence against the direct importance of relative nuclear DNA level to that of organelle DNA synthesis is provided by the observation that the mtDNA of yeast (Williamson et al. 1977) and the cpDNA of C. reinhardtii (Blamire et al. 1974) continue to replicate, at least in the short term, under conditions where nuclear DNA synthesis is inhibited. Unlike the experiments with yeast, however, those with Chlamydomonas failed to show abnormal proportions of cpDNA after inhibition of nuclear DNA synthesis. Whatever the mechanism coordinating the balanced replication of organelle and nuclear DNAs under one set of growth conditions, this coordination appears to be altered under other conditions. For example, the nitrogen starvation procedure used to induce gametogenesis in C. reinhardtii results in cells with half the normal proportion of cpDNA (Sueoka et al. 1967; Wurtz et al. 1977) and at least in some yeast strains, the cellular proportion of mtDNA varies considerably with culture environment (Williamson et al. 1977).
232 Finally, in light: dark synchronized cultures of C. reinhardtii, the presence or absence of acetate in the culture medium can lead to alterations in the temporal pattern of cpDNA synthesis with no effect on the synchronization of cell division per se (Grant et al. 1978).
Acknowledgements. This investigation was supported by Natural Sciences and Engineering Research Council Canada grant no. A-9599 and Dalhousie University Research and Developments Awards to R. W. Lee.
References Behn W, Herrmann RG (1977) Mol Gen Genet 157:25-30 Birky CW Jr (1978) Annu Rev Genet 12:471-512 Blamire J, Fleehtner VR, Sager R (1974) Proc Nat Aead Sci USA 71:2867-2871 Bogenhagen D, Clayton DA (1977) Cell 11:719-727 Chiang KS (1975) Colloques Internationaux du CNRS 240: 147-158 Chiang KS, Sueoka N (1967) Proc Nat Aead Sei US4 57:15061513 Dron M, Robreau G, Le Gal Y (1979) Exp Cell Res 119:301305 Grant D, Swinton DC, Chiang KS (1978) Planta 141:259-267 Grimes GW, Mahler HR, Perlman PS (1974) J Cell Biol 61: 565-574 Howell SH (1972) Nature New Biol 240:264-267
M. Turmel et al.: Chloroplast DNA Synthesis Howell SH, Walker LL (1977) Dev Biol 56:11-23 Kates JR, Chiang KS, Jones RF (1968) Exp Cell Res 49:121135 Lee E-H, Johnson BF (1977) J Bacteriol 129:1066-1071 Lee RW, Jones RF (1973) Mol Gen Genet 121:99-108 Lemieux C, Turmel M, Lee RW (1980) Curr Genet 2:139-147 Lien T, Knutsen G (1979) J Phyeol 15:191-200 Meselson M, Stahl FW (1958) Proe Nat Acad Sci USA 44:671682 Mihara S, Hase E (1971) Plant Celt Physiol 12:225-236 Richards OC, Ryan RS (1974) J Mol Biol 82:57-75 Robreau G, Le Gal Y (1975) Bioehimie 57:703-710 Sager R, Ishida MR (1963) Proe Nat Aead Sei USA 50:725730 Sueoka N, Chiang KS, Kates JR (1967) J Mol Biol 25:47-66 Whiteway MS, Lee RW (1977) Mol Gen Genet 157:11-15 Williamson DH, Fennell DJ (1974) Mol Gen Genet 131:193207 Williamson DH, Johnston LH, Richmond KMV, Game JC (1977) Mitochondrial DNA and the heritable unit of the yeast mitoehondrial genome: a review. In: Bandlow W, Sehwegen RJ, Wolf K, Kandewitz F (eds.) Mitoehondria 1977 Walter de Gruyter Berlin p 1 Wurtz EA, Boynton JE, GiUham NW (1977) Proe Nat Acad Sci USA 74:4552-4556
Communicated by K.P. VanWinkle-Swift Received July 20, 1980
Current Genetics2, 229-232 (1980)
© bySpringer-Verlag 1980
Short Communication Net Synthesis of Chloroplast DNA Throughout the Synchronized Vegetative Cell-Cycle of Chlamydomonas Monique Turmel, Claude Lemieux, and Robert W. Lee Department of Biology,DalhousieUniversity,Halifax,Nova Scotia B3If4JI Canada
Summary. The accumulation of chloroplast and nuclear DNAs during the 12 h light and 12 h dark synchronized vegetative cell-cycle of Chlamydomonas reinhardtii was monitored by the direct optical quantification of these DNAs in the analytical ultracentrifuge. Net synthesis of nuclear DNA was sharply discontinuous and this synthesis occurred during the first 6 h of the dark period. In contrast, the net synthesis of chloroplast DNA appeared continuous throughout the cell-cycle. The rate of this accumulation, however, was greatest in the dark period.
Key words: Chlamydomonas - chloroplast DNA -cellcycle
Introduction Two major DNA components widely differing in base composition are recovered from the unicellular alga C. reinhardtii. One of these, termed a, with a density of 1.723 g/mi, represents most of the cellular DNA and is of nuclear origin (Sueoka et al. 1967; Robreau and Le Gal 1975). The second major DNA component, termed/3, with a density of 1.696 g/ml, is associated with the single chloroplast of this alga (Sager and Ishida 1963; Dron et al. 1979). It usually represents about 10% of the total cellular DNA (Sueoka et al. 1967). This DNA, hereafter called chloroplast DNA (cpDNA), has a molecular weight of 1.34 x 108 daltons (cf. Behn and Herrmann 1977) and the cellular content of cpDNA indicates that there are many copies per haploid vegetative cell. The time course of many cellular events associated with the synchronized vegetative growth of C. reinhardtii Offprint requests to: R. W. Lee
under alternating 12 h light and 12 h dark periods has been well documented (Kates et al. 1968; Mihara and Hase 1971; Howell 1972; Howell and Walker 1977). Cell growth (increment in size) occurs during the light period, while nuclear DNA synthesis, mitosis, and cell division occur in the dark period. The release of daughter cells from the parental cell wall occurs near the end of the dark period or the first hour of the light period. The number of daughter cells produced from a single parent cell (typically 4, 8, or more) is primarily a function of light intensity and hence is influenced by culture density (cf. Lien and Knutsen 1979). Chiang and Sueoka (1967), using l S N J 4 N density transfer techniques similar to those of Meselson and Stahl (1958), examined the pattern and timing of cpDNA replication during the light: dark synchronized vegetative growth of C. reinhardtii. In a cell-cycle during which cell number increased four-fold, most or all cpDNA replication was restricted to the light period as revealed by at least two semiconservative duplications of all cpDNA molecules, while two semiconservative duplications of all nuclear DNA molecules occurred specifically in the dark period. In similar density transfer experiments with synchronized cultures of C. reinhardtii, Lee and Jones (1973) were unable to confirm the classical semiconservative replication pattern for cpDNA as described by Chlang and Sueoka (1967). Rather, after transfer to 14N_ medium only one unimodal cpDNA band was ever visible and this shifted gradually towards lighter densities with continued growth in 14N-medium. Similar replication patterns after density transfer have been reported for the mitochondrial (mt) DNAs of yeast (Williamson and Fennell 1974) and Euglena (Richards and Ryan 1974). More importantly in terms of this communication, the data of Lee and Jones (1973) reveal less synchrony in the cell-cycle timing ofcpDNA replication than described by Chiang and Sueoka (1967). Although the most O172-8083/80/0002/0229/$01.00
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of C. reinhardtii by direct optical quantification of this DNA in the analytical ultracentrifuge. The results, free of ambiguities frequently associated with isotopic labelling experiments, indicate that net cpDNA synthesis is continuous across the cell-cycle.
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Time (hr) Fig. 1. Increase of chloroplast and nuclear DNAs during the synchronized growth of C. reinhardtii (137c, mt+). An 81 culture was synchronized by growth under alternating 12 h light and 12 h dark periods and DNA was extracted and quantified directly in the analytical ultracentrifuge relative to Bacillus subtilis phage SP8 DNA (p = 1.742 g/ml) which was added to the lysates prior to DNA extraction. Details of culture, DNA extraction, and DNA quantification have been described previously (Lemieux et al. 1980). Samples for DNA extraction were taken from volumes of culture containing 2.0-5.0 x 108 cells except that volumes taken from hours-18 and -21 were 2- to 4-times smaller than the volumes taken previously at hour-!2. With the onset of light in cycle-6, the culture density had reached 2.8 x 105 cells/ml. The strain of C. reinhardtii employed here was obtained from Drs. N. W. Gillham and J. E. Boynton who in turn obtained it from Dr. R. P. Levine and hence it appears to be the same one used in the density transfer experiment of Chiang and Sueoka (1967)
dramatic density shifts in cpDNA were detected in the light period, further important shifts were also detected during the dark period but the quantitative significance of these shifts was difficult to access as the cpDNA density approached that of fully 14N-labelled molecules. More recently, Chiang and coworkers (Chiang 1975; Grant et al. 1978) using radioactive DNA precursors have found major labelling of C. reinhardtii cpDNA during both the light and dark phases of synchronized vegetative culture. In an attempt to relate these results to their earlier report (Chiang and Sueoka 1967), it was suggested that the light period incorporation results from net cpDNA synthesis and that repair o f cpDNA may account for the dark period incorporation. In this study we have followed the accumulation of cpDNA during the synchronized vegetative cell-cycle
Results and Discussion Data on the accumulation of cp and nuclear DNAs during the light: dark synchronized growth of C reinhardtii are shown in Fig 1. After five sucessive light: dark cycles, samples were harvested for DNA extraction at various times commencing with the dark period of cycle-6 and ending with the light period of cycle-8, as shown. In cycle-6 and -7, cell number increased 6.9and 3.6-fold during the respective dark periods while nuclear DNA levels, monitored more frequently, showed comparable increases during the first half of these dark periods. In contrast, the accumulation of cpDNA during both the light and dark periods of culture is evident in the samples taken from the dark period of cycle-6 through the dark period of cycle-7. The rate of cpDNA accumulation, however, in both this and other experiments, consistently appears most intense in the dark period during an 8 - 1 0 h interval which lags behind but overlaps with the interval of nuclear DNA synthesis. The period of most intense cpDNA accumulation occurs during the interval of the light: dark cycle when chloroplast division and cytokinesis reportedly take place (Mihara and Hase 1971). The negligible accumulation of cpDNA during the light period of cycle-8 probably resulted from the culture nearing stationary phase. Most cells failed to divide in the dark period which followed. Table 1 reports the average cellular content of total DNA and cpDNA from the culture times of Fig. 1 where cell counts were determined. The percentage of total DNA represented by the cpDNA fraction is reported for all culture times. These data reveal significant cellcycle fluctuations in the percentage of cpDNA as expected from the different cell-cycle synthesis patterns of cp and nuclear DNA, as shown in Fig. 1. Among the cell-cycle times examined, the percentage of cpDNA is maximum (16%-18%) at the end of the light period (hour-12) just before nuclear DNA synthesis and minimum (5%-9%) after three hours of darkness (hour-18) when nuclear DNA synthesis has reduced its proportion. Once again, the hour-12 value of cycle-8 is not representative as the culture was nearing stationary phase. The average cpDNA content at 2 0 - 2 2 x 10 -15 g/cell among cells harvested at the onset of the light period (hour.24/0) is in agreement with previous reports (Lemieux et al. 1980; Whiteway and Lee 1977) and indicates an average cpDNA copy number of about 100 copies per cell. These
M. Turmel et al.: Chloroplast DNA Synthesis
231
Table 1. Cell-cycle changes in total DNA (nuclear DNA plus cpDNA) and cpDNA during the synchronized vegetative growth of C. reinhardtii (from Fig. 1). Determinations of DNA content per eell are corrected for minor cell loses during the cell harvesting and washing steps and epDNA copy number per cell is based on the molecular weight of individual molecules being 134 megadaltons (Behn and Herrmann 1977) Cycle
Timea (h)
Total e p D N A / epDNA DNA/cell cell copies/ (gx1015 ) (gx1015 ) cell
% cpDNA
6 6 6/7 7 7 7 7 7 7/8
12 18 24/0 6 12 15 18 21 24/0
246 185 209 197
8
3
-
-
-
8
12
203
25.1
113
17.5 5.3 10.9 16.3 16.7 11.0 8.6 10.8 11.1 12.8 12.4
43.0 20.2 34.9 22.1
194 91 157 100
Hour 0 = onset of the light period; 6 = midpoint of the light period; 12 - light: dark transition point; 18 = midpoint of the dark period; 24/0 = dark: light transition point (i.e. beginning of a new cycle)
values are undoubtedly lower in the last half of the dark period just after cell division and they obviously increase throughout the light period, reaching maximum in the first half of the dark period just before cell division. Similar experiments with synchronized Chlamydomonas moewusii also reveal net synthesis of cpDNA during both the light and dark phases of culture (unpublished observations) and the cpDNA copy number per cell at, the dark:light transition of culture is also about 10.0 (Lemieux et al. 1980). The data we present on the timing of DNA synthesis during the synchronized vegetative culture of C. reinhardtii are the first in which the changing levels of cp and nuclear DNAs, expressed per ml of culture, were monitored directly by the UV absorption of these DNAs in the analytical ultracentrifuge. Evidence for the efficient extraction of these DNAs which together account for over 96% of the cellular DNA of C reinhardtii (Sueoka et al. 1967) is provided by the correspondence between the combined cp and nuclear DNA levels per cell which we recovered (Table 1), and the range of values reported for whole cell DNA as determined by colorimetric assays with similarly grown cultures (Sueoka e t al. 1967; Whiteway and Lee 1977). Moreover, the temporal pattern of nuclear DNA synthesis as revealed by the approach used in this study is indistinguishable from that obtained by independent methods with similar cultures of this alga (Kates et al. 1968).
The continuous net synthesis of cpDNA which we describe makes it unlikely that each cellular doubling of cpDNA results from a single replication of each individual cpDNA molecule. Assuming that the synthesis of an individual cpDNA molecule occupies only a fraction of the cell-cycle, it is hard to envisage a mechanism by which cells could distinguish those molecules which had already been replicated from those which had not. We, therefore, favor the possibility that the cpDNA molecules of mitotically dividing C. reinhardtii are randomly selected for replication as indicated in studies with the mtDNA of cultured mouse L cells (Bogenhagen and Clayton 1977; see also, Birky 1978). Our inability to discriminate newly replicated from unreplicated cpDNA molecules in density transfer experiments, as mentioned above, makes it extremely difficult to test this hypothesis directly. It is noteworthy that the interval of maximal cpDNA accumulation in C. reinhardtii was detected after the onset of nuclear DNA replication had reduced its proportion to a cell-cycle minimum and that minimal cpDNA accumulation was observed prior to nuclear DNA replication when the proportion of cpDNA had reached a cell-cycle maximum. On the basis of these data, it is tempting to suggest that the rate of cpDNA synthesis occurs in response to a changing cpDNA/nuclear DNA ratio. Such an hypothesis is consistent with the observation that cpDNA content increases with nuclear ploidy in haploid and diploid vegetative cell cultures of C. reinhardtii (Whiteway and Lee 1977). Similar results have been reported for the mtDNA of yeast (Grimes et al. 1974). Alternatively, cell volume, which also increases with nuclear ploidy in both organisms, might be the factor most crucial to the regulation of organelle DNA synthesis (cf. Williamson et al. 1977; Lee and Johnson 1977). Evidence against the direct importance of relative nuclear DNA level to that of organelle DNA synthesis is provided by the observation that the mtDNA of yeast (Williamson et al. 1977) and the cpDNA of C. reinhardtii (Blamire et al. 1974) continue to replicate, at least in the short term, under conditions where nuclear DNA synthesis is inhibited. Unlike the experiments with yeast, however, those with Chlamydomonas failed to show abnormal proportions of cpDNA after inhibition of nuclear DNA synthesis. Whatever the mechanism coordinating the balanced replication of organelle and nuclear DNAs under one set of growth conditions, this coordination appears to be altered under other conditions. For example, the nitrogen starvation procedure used to induce gametogenesis in C. reinhardtii results in cells with half the normal proportion of cpDNA (Sueoka et al. 1967; Wurtz et al. 1977) and at least in some yeast strains, the cellular proportion of mtDNA varies considerably with culture environment (Williamson et al. 1977).
232 Finally, in light: dark synchronized cultures of C. reinhardtii, the presence or absence of acetate in the culture medium can lead to alterations in the temporal pattern of cpDNA synthesis with no effect on the synchronization of cell division per se (Grant et al. 1978).
Acknowledgements. This investigation was supported by Natural Sciences and Engineering Research Council Canada grant no. A-9599 and Dalhousie University Research and Developments Awards to R. W. Lee.
References Behn W, Herrmann RG (1977) Mol Gen Genet 157:25-30 Birky CW Jr (1978) Annu Rev Genet 12:471-512 Blamire J, Fleehtner VR, Sager R (1974) Proc Nat Aead Sci USA 71:2867-2871 Bogenhagen D, Clayton DA (1977) Cell 11:719-727 Chiang KS (1975) Colloques Internationaux du CNRS 240: 147-158 Chiang KS, Sueoka N (1967) Proc Nat Aead Sei US4 57:15061513 Dron M, Robreau G, Le Gal Y (1979) Exp Cell Res 119:301305 Grant D, Swinton DC, Chiang KS (1978) Planta 141:259-267 Grimes GW, Mahler HR, Perlman PS (1974) J Cell Biol 61: 565-574 Howell SH (1972) Nature New Biol 240:264-267
M. Turmel et al.: Chloroplast DNA Synthesis Howell SH, Walker LL (1977) Dev Biol 56:11-23 Kates JR, Chiang KS, Jones RF (1968) Exp Cell Res 49:121135 Lee E-H, Johnson BF (1977) J Bacteriol 129:1066-1071 Lee RW, Jones RF (1973) Mol Gen Genet 121:99-108 Lemieux C, Turmel M, Lee RW (1980) Curr Genet 2:139-147 Lien T, Knutsen G (1979) J Phyeol 15:191-200 Meselson M, Stahl FW (1958) Proe Nat Acad Sci USA 44:671682 Mihara S, Hase E (1971) Plant Celt Physiol 12:225-236 Richards OC, Ryan RS (1974) J Mol Biol 82:57-75 Robreau G, Le Gal Y (1975) Bioehimie 57:703-710 Sager R, Ishida MR (1963) Proe Nat Aead Sei USA 50:725730 Sueoka N, Chiang KS, Kates JR (1967) J Mol Biol 25:47-66 Whiteway MS, Lee RW (1977) Mol Gen Genet 157:11-15 Williamson DH, Fennell DJ (1974) Mol Gen Genet 131:193207 Williamson DH, Johnston LH, Richmond KMV, Game JC (1977) Mitochondrial DNA and the heritable unit of the yeast mitoehondrial genome: a review. In: Bandlow W, Sehwegen RJ, Wolf K, Kandewitz F (eds.) Mitoehondria 1977 Walter de Gruyter Berlin p 1 Wurtz EA, Boynton JE, GiUham NW (1977) Proe Nat Acad Sci USA 74:4552-4556
Communicated by K.P. VanWinkle-Swift Received July 20, 1980
