Cell, Vol. 5, 195-203,

June

1975,

The Temporal

Copyright@

1975

by MIT

Structure

of S Phase

Robert Ft. Klevecz and Beverly A. Keniston Department of Cell Biology Division of Biology City of Hope National Medical Center Duarte, California 91010 Larry L. Deaven Department of Cellular and Molecular Radiobiology University of California Los Alamos Scientific Laboratory Los Alamos, New Mexico 87544

Summary DNA synthesis in the S phase of V79 and CHO Chinese hamster ceils was examined in detail using an automated system for selection and subculturing of mitotic cells and four different assays for DNA synthesis. Flow microfluorometric (FMF) analysis showed that the selected populations were highly synchronous with few noncycling cells. In CHO cells changes in mean and modal fluorescence in the FMF suggested that DNA content increased in a saltatory fashion with lo-20% of the DNA replicated in early S, 40% in mid S, and 40-50% in late S. Pulse labeling and acid precipitation revealed a repeatable pattern of fluctuations in the rate of isotope incorporation with the maximum rate occurring late In S in both V79 and CHO. Autoradiography proved to be the best means of accurately determining the beginning of S phase. Cumulative labeling from mitosis to points in S exaggerated the differences in rate between early and late S, so that significant DNA synthesis in early S might easily be overlooked using this method. In CHO cells DNA-specific fluorescence by the Kissane and Robins assay supported the isotopic incorporation data and the FMF analyses by exhibiting a stepwise increase. In V79 cells, S phase lasts only 5 or 5.5 hr, and consequently the mid S and late S steps in fluorescence are compressed. In V79, greater than 80% of the increase in DNA-specific fluorescence occurred between 4.5 and 7 hr after mitotic selection. In both cell lines, fluorescence in early S phase frequently increased transiently to maximum and then decreased. Introduction In the course of embryogenesis, the length of S phase increases (Remington and Flickinger, 1971), and the chromosomal pattern of replication becomes more complex (Kinsey, 1967; Stambrook and Flickinger, 1970). In early cleavage Drosophila embryos S phase is less than 3 min long, whereas

in the adult somatic cells in culture S phase may take 600 min. This 200 fold increase in the length of time required to replicate the identical genetic complement is somewhat paradoxical, since the rate of movement of the replicative fork appears to be nearly the same in both tissues (Blumenthal, Kriegstein, and Hogness, 1974). Drosophila somatic cells do appear to have fewer initiation sites per length of DNA, but the 5 fold increase in distance between origins observed by Blumenthal et al. (1974) in fiber autoradiographs is still insufficient to account for the greater length of S phase. In mammalian cells in culture, there is a 2 fold increase in the rate of DNA chain growth between early and late S phase, and a 5 fold variation overall (Painter and Schaefer, 1969). Asynchronous DNA replication which was first detected chromosomally (Taylor, 1960) is also manifested temporally in the cell cycle by bursts in tritiated thymidine incorporation within S phase (Klevecz, 1969; Lett and Sun, 1970; Klevecz, Kapp, and Remington, 1974). Synchronous initiation of clusters of replicating units followed by periods of low initiation frequency and diminishing numbers of growing chains has been proposed to explain bursts in 3H-thymidine incorporation (Klevecz and Kapp, 1973). The intermittent replication of such clusters can account for the increased length of S as cells undergo differentiation. The initiation of S phase, the rate of DNA synthesis, and the pattern of DNA accumulation through the cell cycle have been measured and compared using total isotope incorporation from pulse labeling and continuous labeling experiments, percent labeled nuclei in autoradiographs of pulse and continuously labeled synchronous cells, DNA fluorescence/cell using the fluorescent Feulgen assay and the flow microfluorograph (VanDilla et al., 1969) and DNA specific fluorescence/cell using the Kissane and Robins (1958) diaminobenzoic acid assay. Close analysis of the pattern of DNA synthesis in S reveals that DNA content increases in a saltatory fashion, and that the early portion of S phase is a period of low net DNA synthesis which may be mistaken for Gl if methods of measurement other than autoradiography are used. The results presented here may bear on reports of repair replication (Gl thymidine incorporation) in unirradiated cells (Djordjevic et al., 1969) and the failure of DNA synthesis inhibitors such as FUdR, amethopterin, or 2-4 mM thymidine to block cells effectively at the Gl /S boundary (Williams and Cckey, 1970). Results Mitotic cells were selected from cultures growing exponentially in roller bottles by the gentle shearing action of standard growth medium on the surface

Cdl 196

of the monolayer. Cells were selected at constant temperature without the use of altered medium and without the use of inhibitors, since all such treatments can alter normal cellular functions and reduce the number of cells traversing the cycle. The method used here to prepare synchronous V79 and CHO cell cultures should produce minimal perturbation in cellular metabolism. In addition, selection can be optimized in terms of maximum yield of cells consistent with a high mitotic index, and the optimal conditions can be repeatably obtained from selection to selection and from day to day. Initial synchrony (the percentage of cells in mitosis) was always greater than 95% and with V79 and CHO was commonly 98% or 99% (Klevecz, 1975). Viability, and the kinetics of attachment to the substrate were measured for each of a number of repeated selections from a random exponential culture of V79 cells and are shown in Figure 1. Viability, as determined by eosin exclusion, was uniformly high in all cultures as was the ability of each selection to form clones (unpublished observations). The rate of attachment of cells to the surface of the culture vessel following selection was measured as before for WI-38 (Klevecz and Kapp, 1973). Greater than half the cells were attached within 1 hr after selection, and maximum plating was attained by 4 hr. Viability of selected mitotic cells was often better than that of a randomly growing population freshly subcultured 5 hr or 10 hr prior to staining. In V79, the second synchronous metaphase began as an

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Eosin exclusion by V79 cells was measured in each of the selected populations (O-O). The viability of random cultures of cells removed by trypsinization, plated, and scored for viability 5 hr or 10 hr after subculture is also shown (a). Mitotic index was scored as described previously by Remington and Klevecz (1973). The percent viable cells was >96% in all cases. Attachment of the selected cells to the surface of sterile scintillation vials was scored by counting cells/ml in the medium immediately following mitotic selection, and by trypsinization and counting of attached cells at intervals through the cycle (C-O).

abrupt increase in the percent mitotic cells between 8 hr and 9 hr after selection. One intended function for flow microfluorometry was the resolution of age distribution of cells in a randomly growing population (VanDilla et al., 1969). It can also be used to demonstrate quantitatively, the degree of synchrony immediately following mitotic selection and at intervals through the cell cycle. CHO and V79 Chinese hamster cells were stained for DNA content using the acriflavin Feulgen method (Kraemer et al., 1973) and then analyzed at hourly or half-hourly time increments through the cell cycle. The results of this analysis for CHO cells are shown in Figure 2. Between 1 hr and 4 hr after mitotic selection all cells in the population fell into a single symmetrical class with respect to DNA content. Some skewing of the distribution occurred between 5 hr and 6 hr, and by 7 hr a bimodal distribution appeared. The new mode stabilized briefly around channel 50, and then moved out to channel 68. By 12 hr, newly formed daughter cells with the 2C (Gl) DNA content began to appear. Synchrony was maintained well enough so that at 11 hr after selection a population was obtained in which 70% of the cells had the G2 DNA content. The maintenance of such a high degree of synchrony late in the cycle may be due to a combination of high initial synchrony and the constancy of conditions during the selection and subculturing procedure. Temperature was maintained at 37°C throughout all operations. Following selection, the cells were immediately pumped into a culture vessel so that attachment and reentry into Gl began within 5 min after selection. In contrast, manual mitotic selection often involves a considerable number of operations, such as centrifugation, resuspension, and plating, which, even when they are done at ambient temperature, represent a significant time lag and possible perturbation. Immediately after selection and commonly for the ensuing hour, a bimodal distribution of DNA content like that in Figure 3 was detectable. Since the selection procedure removes cells in all stages from prometaphase through early Gl, with the greatest proportion of cells in metaphase, anaphase, or telophase (Klevecz, 1975), it seems likely that the cells with the higher DNA content in Figure 3 represented metaphase (4C) cells and possibly freshly divided doublets which had not yet separated. In some cell lines, particularly V79, there was a tendency for the daughter cells to remain as doublets for a number of hours following selection. These doublets had to be separated prior to fixation and staining in order for accurate values for DNA/cell to be obtained. However, this was not a problem with the CHO cell line, and within the first hour after selec-

Temporal 197

Structure

of S Phase

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Figure 3. Distribution of DNA Synchronous CHO Cells content) Synchronous

40

CHO

CHO cells were synchronized by automated mitotic cell selection as described in the text. Cultures of 1-3 x 107 cells distributed at hourly intervals through the cycle were harvested by trypsinization and fixed for FMF analysis and stained according to the procedure of Kraemer et al. (1973). Wherever possible, equal numbers of cells (50,000 total) were analyzed in the FMF. Modal DNA content/cell of newly divided (Gl) cells is indicated by the dashed line (---). Initial modal channel = 34; modal channel through late Gl and early S = 39-40; modal G2 channel = 68-70.

tion a symmetrical unimodal distribution of cellular DNA content, centering around the 2C value, was obtained, indicating that exclusively Gl cells were present. In the fluorescent Feulgen assay for DNA, complete hydrolysis to produce free deoxyribose must be avoided since, unlike the diphenylamine or Kissane and Robins assays, the DNA content is measured in situ. The possibility must be consid-

Content/Cell

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60

NUMBER from

Mitosis

to G1 in

CHO cells were synchronized as described in the text and stained by the acriflavin-Feulgen method of Kraemer et al. (1973). Only the first 3 hr of the cycle are shown. Freshly detached cells are either in mitosis or are densely staining early Gl cells. The change in condensation states of chromatin is represented schematically in the figures.

ered that this staining procedure may be affected by the availability of the DNA. Experiments such as those shown in Figures 2 and 3 would indicate that the condensation state of the chromatin is a factor in expression of fluorescence. Between 0 hr and 2 hr after mitotic selection, the ceils with a Gl DNA content showed increased fluorescence and moved from channel 21 to 29 in Figure 3 and channel 34 to 39 or 40 in Figure 2. The doublet or 4C population at 1 hr in Figure 3 has been greatly reduced, but the remaining cells show increased fluorescence. That this represented a change in the condensation state of the chromatin and consequently in the accessability of the DNA to the stain seemed likely

Cdl 198

since at this time no labeling of nuclei was detectable by autoradiography. If the shift were due to the condensation state of the chromatin, then it might have been expected to manifest itself as decreased fluorescence in the G2 population (lo-12 hr) as the DNA went from a less condensed to a more condensed state in preparation for division. A small shift, from channel 70 to 68, is apparent in Figure 2, but beyond the interpretable limits of the technique. By 12 hr some cells had completed

the first cycle and had undergone a second division. Most interesting is the fact that the Gl population that reappeared at this time in Figure 2 displayed a modal fluorescence which was equal to that of the telophase doublets in the first generation and not that of the Gl population 2 hr after mitosis. In any case, while the fluorescent Feulgen assay may conceivably be affected by the condensation state of the chromatin, it can at least be used to discriminate between cells in Gl with a 2C DNA

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4. Saltatory

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(a) Comparison of Cell Cycle Parameters of Mitotically Selected CHO and V79 Cells Falcon plastic flasks (75 cm2) were inoculated with 5 X 105 mitotic cells, and 3H-TdR at 5 @ml (50.3 Ci/mmole) was added at the time of inoculation. Labeling was continued through the first 5 hr (V79; open circles) or 9 hr (CHO; closed circles) of the cycle. Cells were trypsinized immediately after the pulse or at half hour intervals through the first 5 hr of the cycle and fixed for autoradiography. Slides were dipped in NTB-10 emulsion and exposed for intervals of 1-8 weeks, Maximum exposure times were judged when longer exposures failed to increase the percentage of labeled cells. Mitotic indices for V79 (-A-A-) and CHO (-A-A-) were determined as described in Figure 1. (b) Rate of Acid-Precipitable Thymidine Incorporation Scintillation vials were inoculated with approximately 1 x 105 CHO cells, and the cultures subjected to the labeling protocol described above. Monolayers were washed and the amount of acid-precipitable 3H-thymidine incorporated into DNA determined as described in the Experimental Procedures section. Total acid precipitable incorporation of thymidine into cells pulse labeled for 30 min at hourly intervals (-A-A-) is shown. (c) DNA-Specific Fluorescence using the Kissane and Robins Assay Mitotic CHO cells were detached at hourly intervals and allowed to progress through the cell cycle. Cultures were harvested and fixed for DABA analysis as described in the Experimental Procedures section. The fluorescence/l03 cells from three replicate cultures at intervals through the cell cycle is shown for two independent experiments. (d) Mean and Modal DNA Content (Channel Number) from an FMF Analysis of Synchronous CHO Cells Data from an experiment like that shown in the frequency distribution in Figure 2 were used to generate mean and modal values for DNA content. In the experiment displayed here, synchronous cultures were prepared at half-hour intervals from 2-8 hr and at hourly intervals thereafter. Major modes (-O-O-); minor modes (0). The major mode is defined as the channel with the greatest number of cells, the minor mode as the channel with a distinct frequency peak having the second greatest number of cells. Mean DNA content/cell is indicated (0) and was determined as described by Klevecz (1975).

Temporal 199

Structure

of S Phase

content and those in G2 with a 4C DNA content, and for our purposes provides a quantitative measure of the degree of synchrony and indicates that only a small fraction of the cells are noncycling. The beginning of S phase was determined by scoring the percent labeled nuclei (PLN) following continuous 3H-thymidine exposure. In Figure 4a, the cell cycle parameters of V79 and CHO are shown together. The DNA synthetic period began 4.5 hr after mitotic selection in CHO and 2.25 hr after selection in V79. The beginning of S cannot be determined using measures such as total tritiated thymidine incorporation or by FMF analysis since in the former case no information on the DNA synthesizing fraction of cells can be obtained, and in the latter the sensitivity is insufficient to accurately score the beginning of S. The fluorescent Feulgen FMF distributions from 5-7 hr after mitosis shown in Figure 2 revealed skewing in the DNA content/cell but no change in the modal value. Between 8 hr and 10 hr, there was a very abrupt shift in the modal value, and it would appear that most cells replicated almost half of their DNA in a relatively restricted portion of S. For convenience in analyzing the data, the mean and modal DNA values derived from data like that in Figure 3 have been plotted in Figure 4d as a function of time after mitotic selection. The values for mean DNA content by FMF analysis showed general agreement with those obtained in Figure 4c using the Kissane and Robins fluorescence assay. In both cases, the increase in fluorescence occurred in a saltatory manner. From a number of experiments in CHO it is concluded that the steps represent an increase in fluorescence of lo-20% between 4 hr and 7 hr, 40% between 7 hr and 9 hr and 40-50% between 9 hr and 11 hr. Fluctuations in the rate of 3H-thymidine incorporation have been reported before for a number of cell lines (Klevecz et al., 1974), and the results of 30 min pulse labeling at hourly intervals through S is shown in Figure 4b to indicate the agreement between thymidine pulse labeling and increases in fluorescence. Most important for the present consideration is the fact that DNA content per cell did not increase appreciably until 6 hr after mitotic selection, although S phase determined by autoradiography began at 4 hr or 4.5 hr. The shift in modal DNA fluorescence, which was a measure of the change in DNA content of the majority of the cells, showed a very abrupt transition between 7 hr and IO hr. An analysis similar to that in Figure 4d was performed on V79 cells and is shown in Figure 5. Mean and modal DNA content increased only slightly during the first half of S phase, but then increased abruptly during late S. The changes in DNA specific fluorescence are similar to those observed using the Kissane and Robins

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DNA

Content

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of Synchro-

V79 cells were synchronized by automated mitotic selection and fixed for FMF analysis at half-hourly or hourly intervals through the cell cycle. The major mode of DNA content is indicated by the closed circles (0-O). The beginning of S phase was determined by autoradiographic analysis (A-A) of the percent labeled nuclei as described in Figure 4.

diaminobenzoic acid (DABA) assay, although the steps in early S phase fluorescence which were quite apparent using the DABA assay (Figure 7) are only marginally visible by acriflavin-Feulgen staining. Since the DABA fluorescence assay has been done much more frequently on closely spaced samples, it is not possible to determine at this time whether more frequent sampling and repeated studies would give a pattern identical to the DABA assay, or whether there are inherent differences in the staining characteristics of the two procedures. The apparent difference between the beginning of S phase and the measurable accumulation of DNA was investigated more directly by comparing four independent measures of DNA synthesis in V79 cells. In Figure 6 cells were labeled either continuously from the time of selection, or pulse labeled 30 min prior to harvest and harvested at 30 min or 60 min intervals through the cycle. The cells were fixed for autoradiography or for total acid precipitable radioactivity counting. Autoradiography is the most sensitive measure of DNA synthesis, since exposure times can be lengthened to permit detection of very low levels of precursor incorporation. S phase was operationally

Cell 200

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HOURS AFTER MITOTIC SELECTION Figure 6. Measuring lative XH-thymidine

S Phase in V79 by Autoradiography Incorporation

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Autoradiographic Estimates of S Phase Falcon plastid flasks (75 cmz) were inoculated with 5 x 105 mitotic V79 cells, and 3H-thymidine at 5 PC/ml (50.3 Ci/mmole) was added either at the time of inoculation (continuous o-0) or at half-hourly intervals thereafter for 30 min (pulse O-O) through the first 5 hr of the cycle. Cells were trypsinized immediately after the pulse or at half hour intervals through the first 5 hr of the cycle and fixed for autoradiography. Incorporation Rate Scintillation vials were inoculated with approximately 1 x 105 cells and the cultures subjected to the labeling protocol described above. Monolayers were washed and the amount of acid precipitable SH-thymidine incorporated into DNA determined using the procedure described in the Experimental Procedures section. Total acid precipitable incorporation of thymidine into cells labeled continuously from mitosis (x-x) and for 30 min at half-hourly intervals (O-O) is shown.

defined by Howard and Pelt (1953) using autoradiographic techniques; hence by definition S phase begins in V79 Chinese hamster cells 2.25 hr after mitotic selection at the time of half maximal PLN. At this time, net thymidine incorporation from continuously administered label appears to be only marginally above background when radioactive incorporation is plotted graphically as a percentage. Incorporation at 2 hr after mitosis equals 5000 cpm as opposed to 95,000 cumulative cpm at 8 hr. Thus incorporation at 2-3 hr represents only 5% of the total and might be dismissed as being due to asynchrony in the population. This is clearly refuted by the curve for labeled nuclei which increases smoothly and with a mid-point at 2.25 hr. If the curve for total thymidine incorporation from continuous labeling were to be extrapolated back to zero as a means of determining the beginning of S phase, S phase would be artefactually displaced two hours into the cell cycle. Continuous labeling appears to exaggerate the differences in synthetic rate between early and late S. In part this may be due to the swelling of endogenous pools at the

Gl /S boundary (Tobey et al., 1974), or possibly the low levels of thymidine kinase present in early S may prevent efficient thymidine utilization (Klevecz, 1969). It may be interesting to examine tritiated thymidine incorporation in cells cultured in a thymidine containing medium. Close analysis using both isotopic incorporation and DNA-specific fluorescence revealed that the rate of DNA synthesis changes markedly in different portions of S phase. Since the changes which occur are rather abrupt, and in the case of V79, closely spaced, frequent sampling is necessary to reveal the discontinuities clearly. In Figure 7, V79 cells were detached at hourly intervals for 5 hr and then at half-hourly intervals for 4 more hr. The last mitotic selection thus contained the youngest cells. DNA content was assayed using DABA fluorescence and compared temporally with the rate of DNA synthesis by a 3H-thymidine incorporation during a 30 min pulse. In early S phase, there was often a transient increase in fluorescence/cell which was concordant with the first maximum in thymidine incorporation rate. The relative decrease in DNA-specific fluorescence after its initial increase varied from a sharp peak as shown in Figure 7, to a simple step increase similar to that shown in Figure 4c for CHO. At 4.5 hr, a second such burst in tritiated thymidine incorporation and fluorescence appears to be superimposed on, and partially obscured by, the third step in synthesis of the bulk of the DNA. The rate of thymidine incorporation increases prior to the accumulation of DNA-specific fluorescence and, in a nonrigorous way, behaves as the first derivative of changing DNA content, as might be expected. Both total thymidine incorporation and DNAspecific fluorescence curves indicate that the bulk of DNA synthesis does not begin in this cell line until 4.5 hr after mitosis or 2 hr after the beginning of S phase. Discussion DNA replication in the mammalian S phase appears to be regulated at the supramolecular level in the sense that discrete bursts in thymidine incorporation and steps in DNA fluorescence occur in a regular temporal pattern within S. This partitioning of families of replicons into early, middle, and late replicating clusters may serve as a coarse control in a hierarchy of controls determining gene expression (Klevecz, 1969; Remington and Klevecz, 1973). The point has also been made that this pattern is most easily detected in diploid or uniform clones of established cell lines and less obvious or absent in heteroploid cells. In this work we have shown by FMF analysis that stepwise increases in DNA-

Temporal 201

Structure

of S Phase

I

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2 Hours Figure

7. DNA-Specific

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8 Selection

Fluorescence

Mitotic cells were detached and allowed to progress through the cell cycle. At 9 hr after the first mitotic selection, all cultures were harvested and fixed for DABA analysis as described in the Experimental Procedures section. The fluorescence004 cells from four replicate scintillation vials at 0.5 hr and 1.0 hr intervals through the cell cycle is shown (upper figure) together with the standard error of the mean. In the lower figure, mitotic cells were detached at 0.5 hr or 1 hr intervals and dispensed into scintillation vials at concentrations of 2.5 x 104 to 1 x 105 cells/vial. Each set of vials (o-o) was labeled for 30 min at hourly or half-hourly intervals as described in Figure 6.

specific fluorescence are expressed by individual cells. The increases in fluorescence by both Feulgen/FMF and the Kissane and Robins DABA assay occur in steps of lo-20%, 40%, and 40-50% representing the early, middle, and late portions of S phase, respectively. The division of S into three distinct time periods does not appear to be an arbitrary consequence of the limited resolution of sampling because cells with S phase lengths from 12

hr down to 5 hr show similar subdivisions (Klevecz et al., 1974). Hand (1975) has used DNA fiber autoradiography to argue that origins which are clustered on the chromosome tend to initiate replication synchronously. The work presented here would tend to affirm this idea. We have frequently observed that the DNA content/cell appears to display a transient increase in early S concomitant with the earliest burst in tritiated thymidine incorporation. This observation is more routinely made when the Kissane and Robins assay is used and V79 cells are analyzed, although it is an apparent, if less obvious feature of DABA fluorescence curves from CHO cells. The possibility is thus raised that there is differential replication and catabolism of a class of DNA in early S. Alternatively, the transient increase in DABA fluorescence, by itself, could represent a change in the availability of the chromatin to undergo the Doebner-von Miller reaction, particularly since the fluorescent Feulgen assay does appear to be affected by the chromatin condensation state. Whatever the cause, it is worth noting that the ability to detect this peak requires frequent sampling of highly synchronous populations of cloned cells with uniform generation times. The differences in the degree of synchrony with which V79 as opposed to CHO cells enter S phase (Figure 4a) may in part explain why the peak is more apparent in the former cell line. Since the transient increase occurs at the beginning of S phase, which begins at 2.25 hr in V79, 4.5 hr in CHO, and 5.5 hr in WI-38 (Klevecz and Kapp, 1973) and not at the M/G1 transition, it would have to be due to an entirely different mechanism from the uncoiling which occurs after mitosis and which apparently affects fluorescent Feulgen staining. The inhibitors, amethopterin, fluorodeoxyuridine (Rueckert and Mueller, 1960), and 2-4 mM thymidine (Xeros, 1962), all act ultimately by depriving the cell of DNA precursors. S phase arrest using amethopterin was applied by Comings and Kakefuda (1968) to cells already synchronized by mitotic selection in an attempt to get more perfect alignment at the GI/S boundary, but more recent work suggests that rather than improving synchrony it may have allowed cells to progress part way into S. Williams and Ockey (1970), using mitotic selection synchrony, found label associated with the nuclear membrane only in late S during the replication of heterochromatin. Amaldi et al. (1972) have shown that DNA synthesis is only partially blocked by FUdR or aminopterin, and Comings and Okada (1973) have shown that cells arrested at the Gl /S boundary using hydroxyurea do not show association of autoradiographic grains with the nuclear membrane. It appears that many nucleotide deprivation methods do not arrest cells at the beginning of S

Cell 202

but rather collect the cells in mid S prior to the replication of bulk DNA and heterochromatin. We suggest that this is due to the fact that early S is a period of low net DNA synthesis during which effective precursor deprivation may be difficult to achieve, possibly because precursor pools swell in preparation for synthesis or because some DNA degradation occurs during this time making thymidylate available for reutilization. In V79 cells autoradiographic analysis of labeled nuclei reveals that S phase begins 2.25 hr after mitosis, while the bulk of the DNA does not begin to accumulate until 6 hr. DNA synthesis inhibitors may block V79 cells at a point 4-5 hr after mitosis and not at the beginning of S. The shortened lengths of S + G2 observed using this method may be a measure of this effect and not a consequence of continued synthesis of RNA and protein required for division as was originally suggested by Rueckert and Mueller (1960). Bostock and Prescott (1971) observed an 8 hr S phase in rabbit endometrium cells by measuring total 3H-TdR incorporation, while noting low levels of incorporation over a considerable portion of the cycle. The labeled cells were detectable autoradiographically, and if scored for percent of labeled nuclei, gave a value for S phase of 18 hr out of a 27 hr cycle. In reporting this finding, the authors suggested that it might be a peculiarity of rabbit cells, since a similar observation had been made by Painter and Schaeffer (1969) also using rabbit cells. The findings reported here and unpublished observations using cultures of Muntiacus muntjac fibroblasts would suggest that low net synthesis in early S is a general occurrence which may be more apparent in diploid early passage cells than it is in established cell lines. Pelt (1971) has used the presence of lightly labeled nuclei in differentiating and possibly nondividing tissues to argue for a class of metabolic DNA. Quite possibly the cells examined in his studies were in a prolonged early S phase. Djordjevic et al. (1969) reported observing spontaneous unscheduled DNA synthesis in Gl HeLa cells, but the method of measuring S phase, total acid precipitable thymidine incorporation does not accurately detect the beginning of S phase. In view of the fact that thymidine incorporation in their study was hydroxyurea sensitive, it seems likely that these workers were examining normal early S phase synthesis and not repair replication. By definition (Howard and Pelt, 1953), S phase is an autoradiographically determined subdivision of the cell cycle. There are many ways to measure DNA synthesis, but only one way to measure S phase.

Experimental

Procedures

Cell Culture V79 and CHO cells were grown and subcultured by a 1:4 or 1:3 split daily in McCoy’s 5a medium containing 20% fetal calf serum and HEPES buffer as described previously (Klevecz et al., 1974). V79 has a narrow modal chromosome number of 19 and a 8.5 hr generation time. CHO has a model chromosome number of 21 and 12.5 hr generation time. At monthly intervals, stock cultures were examined and certified to be free of Mycoplasma contamination using M. pneumonii as a control. In preparation for synchronization, roller bottles were inoculated with 5 x 107 cells 22-24 hr prior to initiation of the mitotic selection. Selected mitotic cells were dispensed into sterile glass scintillation vials or 250 cc Falcon plastic flasks, and cell cycle parameters and measurement of the quality of synchrony were performed as described by Klevecz and Kapp (1973). Thymidine Labeling High specific activity tritiated thymidine (BH-thymidine, 50-60 Ci/ mmole) was purchased from New England Nuclear and was used within a month of the purchase date. Measurement of total 3Hthymidine incorporation was performed using replicate monolayer cultures of V79 growing in scintillation vials. Following exposure to the isotope, or in preparation for DABA fluorescence, monolayers were washed twice in cold Hank’s BSS. Soluble pools were extracted with two 4”C, 10% TCA washes, and the monolayers rinsed in 4°C 10% potassium acetate in 80% ethanol. Cells were then washed once in 95% ethanol at room temperature, once in 2:l ethanol:ether, and once in ether. At this point, the dried cells were either reacted with DABA as described below or counted directly. Upon addition of Aquasol, the dried cell sheet was solubilized. Spectrofluoromeby DNA specific fluorescence was assayed by the method of Kissane and Robins (1958). RNA at concentrations of 0.01 to 10 pg gave no detectable fluorescence above background, while DNA at the same concentrations gave a linear response up to 4900 fluorescence units. Changes in DABA fluorescence in the cell cycle and in CsCl gradients following DNAase digestion. RNAase digestion, and hot acid hydrolysis occurred with the kinetics expected for a DNA-specific reaction (Kapp, Brown, and Klevecz, 1974). Fluorescence was measured in an Aminco-Bowman spectrofluorometer by excitation at 420 nm and emission at 520 nm (uncorrected). The spectrofluorometer was set to zero using 1 N HCI. Extrapolated DNA standard curves, DNAase l-digested cell monolayers and DABA alone all gave similar blank readings. DABA (0.5 ml) at a concentration of 0.4 g/ml was added to each vial and reacted for 45 min at 60°C. Samples were immediately diluted with 2 ml 1 N HCI. The method was originally described by Kissane and Robins (1958) and in a modified form by Hinegardner (1971). Flow Microfluorometry Cells were removed from the flask in trypsin-EDTA and twice centrifuged and resuspended in Hank’s BSS. After the third resuspension at l-4 x IO* cells/ml of BSS, an equal volume of freshly prepared 8% neutral formalin was added to the BSS and mixed gently. Fixed cells were stained with acriflavin-Feulgen as described by Kraemer et al. (1974). Acknowledgment Supported by grants from the National Institute of Child Health and Human Development and from the National Cancer Institute. Portions of the work were performed under the auspices of the US. Atomic Energy Commission. Received

December

16, 1974;

revised

March

28, 1975

Temporal 203

Structure

of S Phase

References Amaldi, F., Carnevali, Cell Res. 74, 367-374. Blumenthal, Cold Spring Bostock,

F., Leoni,

C. J., and Prescott, D. E., and Kakefuda,

Comings,

D. E., and Okada,

Djordjevic, B., Evans, Nature 224, 803-804. R. (1975).

Hinegardner, Howard,

D. S. (1974).

J. Mol. Biol. 60, 151-162.

T. (1968).

J. Mol. Biol. 33, 225-230.

T. A. (1973).

J. Mol. Biol. 75, 609-618.

A. G., and Weill,

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