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Replicon size and rate of fork movement in early S of higher plant cells (Pisum sativum)
Experimental
REPLICON XN EARLY
Cell Research 103 (1976) 395-403
SIZE AND RATE
S OF HIGHER
PLANT
OF FORK CELLS
MOVEMENT (PISUM
SATNUM)
J. VAN’T HOF
Biology Department,Brookhaven National Laboratory,
Upton, NY I1973, USA
SUMMARY Measurements of chromosomal DNA fiber replication of cells of cultured pea root meristems in early S via autoradiography showed a 3-fold increase in rate of fork movement in the first 2 h. The initial rate was 4.5-6 pm h-l but forks active after 90 min moved at nearly 18 pm h-*. The faster movement was not characteristic of all replicons. Certain fibers consisted of replicons of a smaller mean size (38-42 pm) with slowly moving forks (4.5-6 pm h-l fork-‘) and others had replicons almost 50 pm long with forks that moved more rapidly.
The duration of S (T,) of cells of pea root the replicon size of cells as they entered S; meristems either in vivo or in vitro is 4.5- (2) to measure the rate of DNA fiber growth 6.5 h even when subjected to a variety of of cells as they pass from early to mid-S. nutritional manipulations [l-5]. During S, DNA synthesis is not constant. Cells in the MATERIALS AND METHODS first 2 and final hour incorporate [3H]TdR at Tissue culture a lower rate than those in the middle [l]. That neither T, nor the temporal change in The general procedures used for root tip culture, for synchronization of cells at the Gl/S boundary, and for the rate of DNA synthesis are perturbed by nutritional manipulation have been published [l, 2,5]. ionizing radiation, attest to the stability of In brief, to synchronize cells, freshly excised root tips starved of carbohydrate for 48 h to separate the the regulatory factors responsible for T, [2]. were proliferative cells into GI and G2, cultured with 2% Chromosomal DNA fiber growth in pea is sucrose and IO-” M FUdR (S-fluorodeoxyuridine) and [IO-’ M]uridine for 12 h to accumulate cells at the accomplished by approx. 43 000 replicons Gl/S boundary, then cultured for another 12 h in methat function at one time or another during dium without carbohydrate to remove the FUdR, and provided 2% sucrose to begin progression S [6]. In asynchronously dividing cells the finally, through S. Stationary phase (SP) meristems with no average replicon size is 55 pm and that of cells in S were obtained by carbohydrate starvation of roots for 72 h. Cells of these root tips, when cells terminating S is about 38 pm [6, 71. excised provided 2% sucrose, begin DNA synthesis after a Whether or not these smaller replicons also delay of at least 6 h [8]. function when cells first enter S is unknown. However, pea root meristems can Microspectrophotometq of rudei be manipulated nutritionally to produce tis- The cells of stationary phase or synchronized merisue with cells only in the very early por- stems were fixed in ethanol-acetic acid (3 : 1. v/vi. at room temperature for 45 n&r in 5 N tion of S. The experiments described in this hydrolyzed HCI, washed with distilled water, and stained by the paper exploited this fact (1) to determine Feulgen method. The terminal 1.5-2.0 mm tips were
396 (a)
Van’t Hof ---
1
2
3
etc. ---
tengthd
e-----
Midpoint-Midpoint
(b)
--
-.LII----i A
B
cl
C
Center-Center
(4 - _ -
or ..A...
r--
.
.
-
1 I __
.
Center-Center __.__1 7 . ..-.
Fig. I. Line diagrams of three patterns of tandem ar-
rays of grains on autoradiographs of [3H]TdR-labeled chromosomal DNA fibers that describe how rephcon size can be estimated. See Materials and Methods section for further details.
macerated and squashed on microscope slides after the method of Conger & Fairchild [9]. Squash preparations were brought to water and bleached in two successive solutions of 5 g potassium metabisulfite, 50 ml of I N HCI in 1 liter of distilled water for 10 min each, dehydrated through xylene and mounted in Canada balsam. Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei with a Zeiss scanning microspectrophotometer at 560 nm. Extinctions of prophase and half-telophase nuclei were used to normalize the values at 4C and 2C, respectively.
size by dividing the total fiber length by the number of arrays of grains on it. As depicted in fig. 1a, each array represents fiber growth by a fork and because each replicon has two forks the quotient estimates the l/2 replicon size. The sources of error in this measurement stem from inactive forks and from single arrays produced by two forks of the same replicon that have not diverged before the pulse or by the convergence of two forks of adjacent replicons that terminated replication when r3H]TdR was present. The second method used to estimate replicon size utilizes two pairs of tandem arrays of grams arranged on the same fiber. As diagrammed in fig. lb, each pair represents two forks that travel in opposite directions at nearly equal rates. The unlabeled segment between the members of each pair localizes the origin and the distance between the unlabeled midpoints of tandem pairs of arrays approximates the length of DNA fiber between the origins of adjacent replicons. A third measurement utilizes grain arrays of chromosomal DNA fibers that replicate during a step-down pulse protocol. A step-down protocol involves a pulse with high specific activity [3H]TdR followed by a pulse with [3H]TdR of one-third or less the specific activity of the first [lo]. The autoradiograms that result may have contiguous gram density gradients. The higher density being the DNA replicated during the high specific activity pulse and the lower density, the DNA formed when the specific activity was reduced. This method has been described in detail by Huberman & Riggs [ll], Huberman & Tsai [lo], and Callan [12]. The pattern of grain density gradients produced by this protocol is shown diagrammatically (fig. lc). The line drawings assume bidirectional
c
DNA fiber autoradiogsaphy The procedures for autoradiography of chromosomal DNA fibers of pea root cells are published [6]. In the present experiments two changes were introduced: (1) reduction of fixation time with ice-cold buffered 2% formaldehyde from 5-1.5 min; (2) substitution of 3% sarkosyl in 0.05 M EDTA for SDS as the lytic detergent. A shorter fixation time and the use of sarkosyl improved nuclear lysis and provided longer DNA fibers. Measurement of DNA fiber autoradiographs provided two sets of data: the rate of fork movement and the replicon size, i.e., the length of DNA between the origins of two adjacent replicons. The former was obtained from the length of arrays of grains on fibers isolated from cells that were pulsed with C3H]TdR for various durations. Fibers with four or more tandem arrays were selected and approx. 2OWOO arrays were measured for each pulse. Unless noted otherwise, only high specific activity [3H]TdR (50 Ci/mM, 1 mCi/ml) was used. Replicon size was estimated by three methods. The simplest and least accurate measured the l/2 replicon EA-QCell Res 103 (1976)
I 4567
Fig. 2. Abscissa: amount of nuclear DNA (arbitrary unit, U); ordirzafe: no. of nuclei.
Histograms of microspectrophotometric measurements of nuclear DNA content of randomly selected, Feulgen-stained pea root meristem cells expressed in arbitrary units. (a) Nuclei of cells of meristems starved of carbohydrate for 24 h; (b) same as (a) except starvation was 72 h; (c) nuclei of cells of roots that were synchronized at the Gl/S boundary as described in the Materials and Methods. 2C amount, 2.5-4.0 U; 4C amount, 5-g U; 3C (cells in S) 4.0-5.0 U. Each histogram represents measurements of 150 nuclei.
DNA fiber replication
397
centage is less than 1. Microspectrophoto-. metry of nuclei randomly selected for measurement showed 52 9%of the population had a 2C (extinction units 2.54) and 47 9%had a 4C (extinction units 5-g) amount of DNA (fig. 2 rr). The remainder (< 1%) were jn S (extinction units 4-5). Once the cells were arrested the distribution did not change greatly with an a.dditional48 h of starvarion (fig. 2 6). After a total of 72 h approx. 44% were 2C, 55 % were 4C, and < 1% were in S. The data displayed in fig. 2 a, b are from earlier work [7], which also showed that a 16 h treatment of asynchronously dividing pea root meristem cells with lO+ M KJdR produced a tissue with cells still iin S. Kovats & Van? Hof [14] circumvented this problem by simply starving the tissue of carbohydrate for 48 h prior to the use of the inhibitor. Treatment with FUdR of tissue Fr’g. 3. Abscissa: length (pm); ordinare: %. Histograms of the length of arrays of grains on such as that with cell distributions shown in autoradiographs of [3H]TdR labeled chromosomal DNA fibers of SP cells fed 2% sucrose for 6 h and fig. 2a, b can not retain cells in S because then pulsed for different durations. The number of there are none. Once the cells are separated arrays measured, (a) 198; (6) 200; (c) 270; (d) 334. (c) 6OJ50min is pulse time in high then low spec. act. in G1 and G2, the simultaneous provision of [W]TdR; all other pulses (a) 60 min; (b) 90 min; (d) sucrose and FUdR allows cells of the 180 min with high spec. act. only. growth fraction that are in G2 to proceed through mitosis and advance to G1. Meangrowth which is characteristic of pea cells. The length of DNA between the origins of adjacent replicons is estimated from the distance between the central areas of higher grain density (center-center distance). In cases where the initiation of neighboring origins was not simultaneous, a gap results in the central dense portion. This does not impair the measurement, however, because one assumes the origin to be located between the two dense arrays (fig. 1c). The specific activities of the [3H]TdR used in the step-down experiments described in this paper were 50 Ci/mM and 6 Ci/mM; each were used at concentrations of 1 mCi/ml.
RESULTS
40
t
11, 30
The distribution of cells irz the cycle Earlier experiments showed that approx. 20% of the cells are synthesizing DNA in an asynchronously dividing pea root meristem [13]. After 24 h of starvation the per-
I 60
90
120
11 150
I80
Fig. 4. Abscissa: [SH]TdR pulse (min); ordinate: average length arrays (pm). The average length of arrays of grains on autoradiographs of chromosomal DNA fibers of cells as they enter S expressed as a function of [W]TdR pulse. 0, SP cells fed 2% sucrose for 6 h; 0. cells synchronized at the Cl/S boundary.
398
Van’t Hof
radiographs of [3H]TdR-labeled chromosomal DNA fibers of SP ce!.ls fed 2% sucrose for 10 h before the isotope pulse. Arrays produced by a step-down pulse
of 4.5 min high, 90 min low spec. act. (a), (b), (d), (e) and v); arrays shown in (c) from a step-down pulse of 60 min high, 60 min low spec. act. [3H]TdR. Bar, 50 pm.
while, the cells in Gl are prevented from progression to S. Tissue treated in this manner had the cell distribution shown in fig. 2c. As indicated by microspectrophotometry, 70 % of the cells were 2C, 29 % were 4C, and 1% were 2-4C. The 3C cells were not in all portions of S but were grouped at the very end of DNA synthesis. The cytokinetic experiments of Kovacs & Van? Hof
[14] showed that the cells grouped at the Cl/S boundary are viable and capable of at least one complete division cycle. Also, because T, of the synchronized cells is approx. 5.5 h, it is no different from that of asynchronously dividing pea root meristem cells [.5]. The similarity of T, as well as the cycle duration of cells treated with FUdR in the manner described above with that of
Fig. 5. Photomicrographs of arrays of grains on auto-
Exp Cell Res 103 (1976)
DNA fiber replication
r
F, I 90 20 30 40 50 60 70 80 LOO 110 (a) length (pm); (b) l/2 rep (pm); (c) midpoint-midpoint distance (pm); ordinate: ‘%. Histograms of measurements from autoradiographs of chromosomal DNA fibers with short, uniform tandem arrays of grains from SP cells fed sucrose 10 h and then pulsed with [3H]TdR in a step-down protocol of 45 min high, 90 min low spec. act. (a) Length of arrays of grains; (b) l/2 replicon size; (c) midpointmidpoint distance between tandem symmetrical pairs of arrays. The number measured (a) 355; (b) 100; (c) 121.
399
that averaged 4.5 pm in length and ranged from 2 to 20 pm (fig= 3a). The average length of fiber synthesized during a 90 mm pulse was 6.8 pm (fig. 3 b) and pulses of 120 min (60 min high and 60 min low spec. act.) and 180 min resulted in arrays of grains that averaged 13.8 and 32 vrn, respectively (fig. 3c, d). With longer [3H]Td both the mean and the range of sizes of fiber replicated increased. At 90 mm, segments varied in size from 2 to 25 pm, at 120 min from 2 to 80, and finally, labeled fibers of 5 to more than 150 pm were measured after a pulse of 180 min. A comparison of the length of DNA replicated during pulses of different durations revealed that fork movement was not constant. When the
40
Fig. 6. Abscissa:
” *’ ”
cells never treated with the inhibitor shows that the lethal inhibition of FUdR as described by Ockey [15] for mammalian cells does not apply in the present case. Rate offork movement of SP cells fed carbohydrate
Six hours after provision of 2% sucrose to SP meristems, the first few cells cross the Gl/S boundary, begin to synthesize DNA, and progress through S. The length of DNA replicated at this time was measured by pulses of [3H]TdR of 60-180 mm duration. A 60 min pulse produced arrays of grains
7. Abscissa: (a) length @m); (b) center-center distance (pm); ordinate: ‘3%. Histograms of measurements from autoradiographs of chromosomal DNA fibers with long gram density gradients from SP cells fed sucrose 10 h then pulsed with [3H]TdR in a step-down protocol of 45 min high, 90 mm low spec. act. (a) Length of the arrays of grains; (b) center-center distance between adjacent replicons. Number of measurements (a) 216; (b) 253.
Fig.
400
Van’t Hof
Table 1. The average length of DNA replicated (pm) and the average replicon size (pm) offibers with symmetrical tandem pairs of arrays of nearly uniform length (A) and offibers with asymmetrical tandem arrays of varied lengths (B) of SP cells fed carbohydrate for 6 or 10 h before labeling with r3H]TdR Fiber class Sucrose incubation (hours)
A
B
Length
Size
Length
Size
Pulse
6 10
6.8 7
38 39
12.7 18
49
90 min high spec. act. 45 min high, 90 min low spec. act.
average length was expressed as a function of the duration of the pulse, a complex curve resulted (fig. 4, solid circles). Initially it had a slope that corresponded to a rate of fork movement of 5-6 pm h-l and later on, with longer pulses, the rate increased to nearly 18 pm h-l. Rate offork movement of cells synchronized at the GlIS boundary
The 3-fold change in the rate of DNA fiber growth of SP cells was verified with cells synchronized at the Gl/S boundary. In these experiments the high specific activity pulses were given to cells 30 min after they were provided sucrose. Their temporal position in S at the termination of the pulse was 30 min plus the duration of the r3H]TdR label. When the average length of DNA replicated by these cells was plotted as a function of the duration of the r3H]TdR pulse a curve resulted that had an initial slope of 5-6 ,um h-l (fig. 4). Later, with longer pulses of 60 and 90 min, the slope increased to almost 18 pm h-l. It is apparent that the curves in fig. 4 representing SP cells fed carbohydrate and cells synchronized at the Gl/S boundary are nearly identical. Both show that the rate of DNA fiber growth at the beginning of S is one-third E.rp CeNRes 103 (1976)
that of cells that have completed 90 min or more of DNA replication. Replicons
that fimction
in early S
There are three plausible explanations for the 3-fold change in the rate of DNA fiber growth: (1) The change reflects the movement of forks that begin replication slowly and then later move more rapidly; (2) the different rates are properties of certain replicons that begin DNA fiber growth at different times in S; (3) the replicons of different cell subpopulations become distinctive as a result of differentiation, as shown by Callan [12]. Of these three possibilities, the first two were testable within the context of the present experiments. Therefore, SP meristems were fed 2 % sucrose for 10 h and then pulsed first for 45 min with high and then 90 min with low specific activity r3H]TdR. These meristems have cells just beginning DNA synthesis, a few in mid-S, but none in late S [16]. Upon examination of the autoradiographs of labeled DNA fibers, it became apparent that there were two distinct patterns of tandem arrays of grains. One was formed by relatively long arrays and many of these had tails of contiguous grain density gradients indicative of bidirectional fork movement (fig. 5a, d, e).
DNA fiber replication
The other pattern consisted of short tandem arrays of high grain density (fig. 5b, c, f). Fibers with one pattern or the other often were grouped on the microscope slide making selective measurements possible. One could determine both the rate of fork movement and the replicon size on the same fibers. The histograms shown in fig. 6 are data obtained from fibers with patterns of short, dense arrays of grains, such as those shown in fig. 5 b, c, $ The average length of these arrays was 7 pm and they ranged in size from 3-20 pm. However, most of the replicated segments, 78 %, were in the 5-10 pm category (fig. 6~2). The rate of DNA fiber growth was 3.1 pm h-l fork-l and the replicon size estimated from l/2 replicon lengths was 39 pm (fig. 6b). A slightly larger size was obtained from measurements of the midpoint-midpoint distances between sequential pairs of tandem arrays. These measurements had a mean of 42 pm and a mode of 35-40 pm (fig. 6~); slightly more than 22 % of the replicons were larger than 50 pm. Fibers with the longer array patterns (fig. 5a, d, e) showed grain density gradients that averaged 18 pm in length (fig. 7a). These had a mode of 10-15 pm and ranged from 3-80 pm. The average rate of fork movement was 8 pm h-l and the replicon size estimated from center-center distances was 49 pm. The shortest replicon measured was 15 pm and the longest was 125 pm. The modal replicon size was 3MO pm and approx. 40% of these were more than 50 pm long (fig. 7 b). To verify that cells in very early S have chromosomal DNA fibers with two distinctive replicon classes, midpoint-midpoint measurements were made on fibers of SP cells fed sucrose 6 h prior to a 90 min pulse with [3H]TdR. From these measurements
40
the average replicon size was estimated to be 38 pm, the modal size was 35-40 pm and 23 % were longer than 50 pm. The average rate of fiber growth was 4.5 pm h-’ fork-‘. While many of the DNA fibers had repiicons with characteristics that were consistent with those expected of smaller units with slowly moving forks, there were others that had asymmetrical arrays of varied lengths (3-55 km), i.e., there were fibers with tandem arrays of different lengths and the various arrays were not ordered in the fashion described in fig. 1b. On the fibers with less uniform arrays the average rate of movement/fork was 8.5 pm h-” and the average length of labeled DNA was 12.7 pm. The presence of these faster forks indicated that activation of replication of both classes of replicons occurred within a reiatively short time in early S. In table I are data from SP cells incubated 6 or IO sucrose. It is recognized that some of the slower moving forks only appear as such because they are of replicons that were activated during the latter portion of the pulse. However, it is unlikely that all fall into this category since the density of the grain arrays is more indicative of activity during the high specific activity pulse than during the time when the isotope was more dilute. A more attractive interpretation is that the slower forks maintain a constant pace as the cells progress through S. DISeUSSION Temporal changes in rate offork movement in S
In some plant and animal cells DNA replication is initiated by replicons in which the rate of fork movement is lower in the early portion of S than when the cells are in midor late S. For example, HeLa cells at the beginning and at the end of S have fork
402
Van’t Hof
age nuclei have replicons of approx. 3 pm movement rates of 18-30 and 66 pm h-l, respectively [17]. Also, Chinese hamster that are shorter than those of cultured cells, ovary cells in early S have forks that move but the cultured cells themselves have two 12.6 pm h-l on the average, while those ac- size groups, one of about 9 and another of tivated 7 h later move 38 pm h-l [18]. In 19 pm [21]. Also, in one of two species of another experiment with Chinese hamster Dipidomys, D. ordii, there is a group of cells the rate increased 2.6 times within a fibers with unusual replication patterns 4 h period as the cells passed from early to [22]. However, the size of these replicons is late S [18]. In both experiments the in- difficult to ascertain because of the manner creased fork rate occurred after 1 h or more. in which the measurements were made [23]. If the center-to-center distances were In pea cells the average rate of replication in very early S was about 6 while that of measured as described in ref. [23], then the sizes reported represent the distance becells that were/4,Omin into S was 18 pm h-l (fig. 4). That still higher rates of movement tween the two forks of the same replicon occur in pea in mid- or late-S is evident from rather than between two adjacent origins the fact that the average rate measured in of neighboring replicons. The two size classes of replicons in pea asynchronously dividing cells was 29 pm h-l [6]. However, precisely when in S the may be significant for three reasons, each rapid forks are activated in pea remains to of which is not exclusive of the other. (1) It is possible that the smaller replicons are be determined. The average replicon size of chromo- located on certain DNA fibers that represomal DNA fibers of pea cells at the very sent a normal part of the total genome that beginning of S and of cells that are ter- is replicated from early to late S. Assuming minating replication is 38-42 pm. This value that each fork moved at a constant rate of is 15 pm shorter than the average size meas- about 6 pm h-l, 34 h would be needed to ured from asynchronously dividing pea root replicate 38-42 pm of DNA. Thus, these cells (average size 55 pm). Approx. 20% of smaller replicons, with slowly moving the replicons of asynchronous cells are forks, are active during 60-90 % of T, and may be the major element in the determina40 pm or less and of these 15% are 35 40 pm long [6]. Also, fibers with short ar- tion of its magnitude. (2) It is likely that the smaller replicons rays that are closely spaced, as shown in fig. 5b, c, f, are found on autoradiographs reflect synthetic activity of a subpopulation of DNA of cells that never were starved or of cells within the root that are a part of treated with FUdR (see fig. 11 of ref. [6]). the growth fraction but are also differentiated anatomically within the root. If this Significance of different replicon were the case, a change in the replicon size sizes is associated with cell differentiation, as Before discussing the possible significance suggested by Callan [ 191. of replicon size classes, it should be pointed (3) The smaller replicons may have been out that cells of amphibians and insects induced by carbohydrate starvation. While have at least two size classes that are more this notion is applicable to starved cells, frequently observed. In species of Triturus it is not supported by the fact that cells never starved also have DNA fibers with a the size is different when cells differentiate [ 19,201 and in Drosophila not only do cleav- smaller replicon size class [6]. Exp CellRes 103(1976)
DNA fiber replication
As mentioned earlier, Hori & Lark [22] observed DNA fibers with replicons that had an unusual replication pattern in one of two species of Dipidomys. Species specificity, consequently, is a factor to consider when discussing replication patterns. Therefore, all inferences and interpretations pertaining to the patterns of pea DNA should be restricted to this species until further detailed data are obtained of DNA fibers of other plants. The author wishes to acknowledge the technical assistance of MS C. Bjerknes and Mr J. Clinton, both of whom along with Dr A. Kuniyuki contributed constructively to the ideas expressed in the paper. Research carried out at Brookhaven National Laboratory under the auspices of the US Energy Research and Development Administration.
REFERENCES 1. Kovacs, C J & Van? Hof, J, Radiat res 48 (1971) 95. 2. Van’t Hof, J, Advances in radiation research (ed J F Duplan & A Chapiro) vol. 2, p. 881. Gordon & Breach Science Publ., New York (1973).
403
Van’t Hof, J, Radiat res 41 (1970) 538. - Exp cell res 37 (1965) 292. -J cell bio127 (1965) 179. - Exp cell res 93 (1975) 95. -Ibid 99 (1976) 47. Webster, P L & Van’t Hof, J, Am j bot 57 (1970) 130. 9. Conger, A D & Fairchild, L M, Stain technol 28 (1953) 281. 10. Huberman, J A & Tsai. A J, 5 mol biol 75 (1973) 5. 11. Huberman, J A & Riggs, A D, J mol biol32 (1968) 327. 12. Callan, H G, Brit med bull 29 (1973) 192. 13. Van’t Hof, J, Brookhaven symp bio125 (1973) 152. 14. Kovacs, C J & Van? Hof, J, J cell biol 47 (1970) 536. 15. Ockev, C H, EXD cell res 70 (1972) 203. 16. Van’tHof, J, J cell bio137 (1968) 773. 17. Painter, R B & Schaefer, A W, J mol biol 58 (1971) 289. 18. Housman. D & Huberman. J A. J mol biol. 94 (1975) 173. 19. Callan. H G. Cold Sorine Harbor svmo I L.want bioi 38 (1974) 195, - 20. Tavlor. J H. Int rev cvtol37 (1974) 1. 21. Blumenthal; A B, Kridgstein,‘H J h Hogness, D S. Cold Spring Harbor symp quant bio138 (1974) 205. 22. Hori, T & Lark, K G, J mol biol88 (1974) 221. 23. - Ibid 77 ( 1973)391. 3. 4. 5. 6. 7. 8.
Received June 2 1, 1976 Accepted July 7, 1976
REPLICON XN EARLY
Cell Research 103 (1976) 395-403
SIZE AND RATE
S OF HIGHER
PLANT
OF FORK CELLS
MOVEMENT (PISUM
SATNUM)
J. VAN’T HOF
Biology Department,Brookhaven National Laboratory,
Upton, NY I1973, USA
SUMMARY Measurements of chromosomal DNA fiber replication of cells of cultured pea root meristems in early S via autoradiography showed a 3-fold increase in rate of fork movement in the first 2 h. The initial rate was 4.5-6 pm h-l but forks active after 90 min moved at nearly 18 pm h-*. The faster movement was not characteristic of all replicons. Certain fibers consisted of replicons of a smaller mean size (38-42 pm) with slowly moving forks (4.5-6 pm h-l fork-‘) and others had replicons almost 50 pm long with forks that moved more rapidly.
The duration of S (T,) of cells of pea root the replicon size of cells as they entered S; meristems either in vivo or in vitro is 4.5- (2) to measure the rate of DNA fiber growth 6.5 h even when subjected to a variety of of cells as they pass from early to mid-S. nutritional manipulations [l-5]. During S, DNA synthesis is not constant. Cells in the MATERIALS AND METHODS first 2 and final hour incorporate [3H]TdR at Tissue culture a lower rate than those in the middle [l]. That neither T, nor the temporal change in The general procedures used for root tip culture, for synchronization of cells at the Gl/S boundary, and for the rate of DNA synthesis are perturbed by nutritional manipulation have been published [l, 2,5]. ionizing radiation, attest to the stability of In brief, to synchronize cells, freshly excised root tips starved of carbohydrate for 48 h to separate the the regulatory factors responsible for T, [2]. were proliferative cells into GI and G2, cultured with 2% Chromosomal DNA fiber growth in pea is sucrose and IO-” M FUdR (S-fluorodeoxyuridine) and [IO-’ M]uridine for 12 h to accumulate cells at the accomplished by approx. 43 000 replicons Gl/S boundary, then cultured for another 12 h in methat function at one time or another during dium without carbohydrate to remove the FUdR, and provided 2% sucrose to begin progression S [6]. In asynchronously dividing cells the finally, through S. Stationary phase (SP) meristems with no average replicon size is 55 pm and that of cells in S were obtained by carbohydrate starvation of roots for 72 h. Cells of these root tips, when cells terminating S is about 38 pm [6, 71. excised provided 2% sucrose, begin DNA synthesis after a Whether or not these smaller replicons also delay of at least 6 h [8]. function when cells first enter S is unknown. However, pea root meristems can Microspectrophotometq of rudei be manipulated nutritionally to produce tis- The cells of stationary phase or synchronized merisue with cells only in the very early por- stems were fixed in ethanol-acetic acid (3 : 1. v/vi. at room temperature for 45 n&r in 5 N tion of S. The experiments described in this hydrolyzed HCI, washed with distilled water, and stained by the paper exploited this fact (1) to determine Feulgen method. The terminal 1.5-2.0 mm tips were
396 (a)
Van’t Hof ---
1
2
3
etc. ---
tengthd
e-----
Midpoint-Midpoint
(b)
--
-.LII----i A
B
cl
C
Center-Center
(4 - _ -
or ..A...
r--
.
.
-
1 I __
.
Center-Center __.__1 7 . ..-.
Fig. I. Line diagrams of three patterns of tandem ar-
rays of grains on autoradiographs of [3H]TdR-labeled chromosomal DNA fibers that describe how rephcon size can be estimated. See Materials and Methods section for further details.
macerated and squashed on microscope slides after the method of Conger & Fairchild [9]. Squash preparations were brought to water and bleached in two successive solutions of 5 g potassium metabisulfite, 50 ml of I N HCI in 1 liter of distilled water for 10 min each, dehydrated through xylene and mounted in Canada balsam. Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei with a Zeiss scanning microspectrophotometer at 560 nm. Extinctions of prophase and half-telophase nuclei were used to normalize the values at 4C and 2C, respectively.
size by dividing the total fiber length by the number of arrays of grains on it. As depicted in fig. 1a, each array represents fiber growth by a fork and because each replicon has two forks the quotient estimates the l/2 replicon size. The sources of error in this measurement stem from inactive forks and from single arrays produced by two forks of the same replicon that have not diverged before the pulse or by the convergence of two forks of adjacent replicons that terminated replication when r3H]TdR was present. The second method used to estimate replicon size utilizes two pairs of tandem arrays of grams arranged on the same fiber. As diagrammed in fig. lb, each pair represents two forks that travel in opposite directions at nearly equal rates. The unlabeled segment between the members of each pair localizes the origin and the distance between the unlabeled midpoints of tandem pairs of arrays approximates the length of DNA fiber between the origins of adjacent replicons. A third measurement utilizes grain arrays of chromosomal DNA fibers that replicate during a step-down pulse protocol. A step-down protocol involves a pulse with high specific activity [3H]TdR followed by a pulse with [3H]TdR of one-third or less the specific activity of the first [lo]. The autoradiograms that result may have contiguous gram density gradients. The higher density being the DNA replicated during the high specific activity pulse and the lower density, the DNA formed when the specific activity was reduced. This method has been described in detail by Huberman & Riggs [ll], Huberman & Tsai [lo], and Callan [12]. The pattern of grain density gradients produced by this protocol is shown diagrammatically (fig. lc). The line drawings assume bidirectional
c
DNA fiber autoradiogsaphy The procedures for autoradiography of chromosomal DNA fibers of pea root cells are published [6]. In the present experiments two changes were introduced: (1) reduction of fixation time with ice-cold buffered 2% formaldehyde from 5-1.5 min; (2) substitution of 3% sarkosyl in 0.05 M EDTA for SDS as the lytic detergent. A shorter fixation time and the use of sarkosyl improved nuclear lysis and provided longer DNA fibers. Measurement of DNA fiber autoradiographs provided two sets of data: the rate of fork movement and the replicon size, i.e., the length of DNA between the origins of two adjacent replicons. The former was obtained from the length of arrays of grains on fibers isolated from cells that were pulsed with C3H]TdR for various durations. Fibers with four or more tandem arrays were selected and approx. 2OWOO arrays were measured for each pulse. Unless noted otherwise, only high specific activity [3H]TdR (50 Ci/mM, 1 mCi/ml) was used. Replicon size was estimated by three methods. The simplest and least accurate measured the l/2 replicon EA-QCell Res 103 (1976)
I 4567
Fig. 2. Abscissa: amount of nuclear DNA (arbitrary unit, U); ordirzafe: no. of nuclei.
Histograms of microspectrophotometric measurements of nuclear DNA content of randomly selected, Feulgen-stained pea root meristem cells expressed in arbitrary units. (a) Nuclei of cells of meristems starved of carbohydrate for 24 h; (b) same as (a) except starvation was 72 h; (c) nuclei of cells of roots that were synchronized at the Gl/S boundary as described in the Materials and Methods. 2C amount, 2.5-4.0 U; 4C amount, 5-g U; 3C (cells in S) 4.0-5.0 U. Each histogram represents measurements of 150 nuclei.
DNA fiber replication
397
centage is less than 1. Microspectrophoto-. metry of nuclei randomly selected for measurement showed 52 9%of the population had a 2C (extinction units 2.54) and 47 9%had a 4C (extinction units 5-g) amount of DNA (fig. 2 rr). The remainder (< 1%) were jn S (extinction units 4-5). Once the cells were arrested the distribution did not change greatly with an a.dditional48 h of starvarion (fig. 2 6). After a total of 72 h approx. 44% were 2C, 55 % were 4C, and < 1% were in S. The data displayed in fig. 2 a, b are from earlier work [7], which also showed that a 16 h treatment of asynchronously dividing pea root meristem cells with lO+ M KJdR produced a tissue with cells still iin S. Kovats & Van? Hof [14] circumvented this problem by simply starving the tissue of carbohydrate for 48 h prior to the use of the inhibitor. Treatment with FUdR of tissue Fr’g. 3. Abscissa: length (pm); ordinare: %. Histograms of the length of arrays of grains on such as that with cell distributions shown in autoradiographs of [3H]TdR labeled chromosomal DNA fibers of SP cells fed 2% sucrose for 6 h and fig. 2a, b can not retain cells in S because then pulsed for different durations. The number of there are none. Once the cells are separated arrays measured, (a) 198; (6) 200; (c) 270; (d) 334. (c) 6OJ50min is pulse time in high then low spec. act. in G1 and G2, the simultaneous provision of [W]TdR; all other pulses (a) 60 min; (b) 90 min; (d) sucrose and FUdR allows cells of the 180 min with high spec. act. only. growth fraction that are in G2 to proceed through mitosis and advance to G1. Meangrowth which is characteristic of pea cells. The length of DNA between the origins of adjacent replicons is estimated from the distance between the central areas of higher grain density (center-center distance). In cases where the initiation of neighboring origins was not simultaneous, a gap results in the central dense portion. This does not impair the measurement, however, because one assumes the origin to be located between the two dense arrays (fig. 1c). The specific activities of the [3H]TdR used in the step-down experiments described in this paper were 50 Ci/mM and 6 Ci/mM; each were used at concentrations of 1 mCi/ml.
RESULTS
40
t
11, 30
The distribution of cells irz the cycle Earlier experiments showed that approx. 20% of the cells are synthesizing DNA in an asynchronously dividing pea root meristem [13]. After 24 h of starvation the per-
I 60
90
120
11 150
I80
Fig. 4. Abscissa: [SH]TdR pulse (min); ordinate: average length arrays (pm). The average length of arrays of grains on autoradiographs of chromosomal DNA fibers of cells as they enter S expressed as a function of [W]TdR pulse. 0, SP cells fed 2% sucrose for 6 h; 0. cells synchronized at the Cl/S boundary.
398
Van’t Hof
radiographs of [3H]TdR-labeled chromosomal DNA fibers of SP ce!.ls fed 2% sucrose for 10 h before the isotope pulse. Arrays produced by a step-down pulse
of 4.5 min high, 90 min low spec. act. (a), (b), (d), (e) and v); arrays shown in (c) from a step-down pulse of 60 min high, 60 min low spec. act. [3H]TdR. Bar, 50 pm.
while, the cells in Gl are prevented from progression to S. Tissue treated in this manner had the cell distribution shown in fig. 2c. As indicated by microspectrophotometry, 70 % of the cells were 2C, 29 % were 4C, and 1% were 2-4C. The 3C cells were not in all portions of S but were grouped at the very end of DNA synthesis. The cytokinetic experiments of Kovacs & Van? Hof
[14] showed that the cells grouped at the Cl/S boundary are viable and capable of at least one complete division cycle. Also, because T, of the synchronized cells is approx. 5.5 h, it is no different from that of asynchronously dividing pea root meristem cells [.5]. The similarity of T, as well as the cycle duration of cells treated with FUdR in the manner described above with that of
Fig. 5. Photomicrographs of arrays of grains on auto-
Exp Cell Res 103 (1976)
DNA fiber replication
r
F, I 90 20 30 40 50 60 70 80 LOO 110 (a) length (pm); (b) l/2 rep (pm); (c) midpoint-midpoint distance (pm); ordinate: ‘%. Histograms of measurements from autoradiographs of chromosomal DNA fibers with short, uniform tandem arrays of grains from SP cells fed sucrose 10 h and then pulsed with [3H]TdR in a step-down protocol of 45 min high, 90 min low spec. act. (a) Length of arrays of grains; (b) l/2 replicon size; (c) midpointmidpoint distance between tandem symmetrical pairs of arrays. The number measured (a) 355; (b) 100; (c) 121.
399
that averaged 4.5 pm in length and ranged from 2 to 20 pm (fig= 3a). The average length of fiber synthesized during a 90 mm pulse was 6.8 pm (fig. 3 b) and pulses of 120 min (60 min high and 60 min low spec. act.) and 180 min resulted in arrays of grains that averaged 13.8 and 32 vrn, respectively (fig. 3c, d). With longer [3H]Td both the mean and the range of sizes of fiber replicated increased. At 90 mm, segments varied in size from 2 to 25 pm, at 120 min from 2 to 80, and finally, labeled fibers of 5 to more than 150 pm were measured after a pulse of 180 min. A comparison of the length of DNA replicated during pulses of different durations revealed that fork movement was not constant. When the
40
Fig. 6. Abscissa:
” *’ ”
cells never treated with the inhibitor shows that the lethal inhibition of FUdR as described by Ockey [15] for mammalian cells does not apply in the present case. Rate offork movement of SP cells fed carbohydrate
Six hours after provision of 2% sucrose to SP meristems, the first few cells cross the Gl/S boundary, begin to synthesize DNA, and progress through S. The length of DNA replicated at this time was measured by pulses of [3H]TdR of 60-180 mm duration. A 60 min pulse produced arrays of grains
7. Abscissa: (a) length @m); (b) center-center distance (pm); ordinate: ‘3%. Histograms of measurements from autoradiographs of chromosomal DNA fibers with long gram density gradients from SP cells fed sucrose 10 h then pulsed with [3H]TdR in a step-down protocol of 45 min high, 90 mm low spec. act. (a) Length of the arrays of grains; (b) center-center distance between adjacent replicons. Number of measurements (a) 216; (b) 253.
Fig.
400
Van’t Hof
Table 1. The average length of DNA replicated (pm) and the average replicon size (pm) offibers with symmetrical tandem pairs of arrays of nearly uniform length (A) and offibers with asymmetrical tandem arrays of varied lengths (B) of SP cells fed carbohydrate for 6 or 10 h before labeling with r3H]TdR Fiber class Sucrose incubation (hours)
A
B
Length
Size
Length
Size
Pulse
6 10
6.8 7
38 39
12.7 18
49
90 min high spec. act. 45 min high, 90 min low spec. act.
average length was expressed as a function of the duration of the pulse, a complex curve resulted (fig. 4, solid circles). Initially it had a slope that corresponded to a rate of fork movement of 5-6 pm h-l and later on, with longer pulses, the rate increased to nearly 18 pm h-l. Rate offork movement of cells synchronized at the GlIS boundary
The 3-fold change in the rate of DNA fiber growth of SP cells was verified with cells synchronized at the Gl/S boundary. In these experiments the high specific activity pulses were given to cells 30 min after they were provided sucrose. Their temporal position in S at the termination of the pulse was 30 min plus the duration of the r3H]TdR label. When the average length of DNA replicated by these cells was plotted as a function of the duration of the r3H]TdR pulse a curve resulted that had an initial slope of 5-6 ,um h-l (fig. 4). Later, with longer pulses of 60 and 90 min, the slope increased to almost 18 pm h-l. It is apparent that the curves in fig. 4 representing SP cells fed carbohydrate and cells synchronized at the Gl/S boundary are nearly identical. Both show that the rate of DNA fiber growth at the beginning of S is one-third E.rp CeNRes 103 (1976)
that of cells that have completed 90 min or more of DNA replication. Replicons
that fimction
in early S
There are three plausible explanations for the 3-fold change in the rate of DNA fiber growth: (1) The change reflects the movement of forks that begin replication slowly and then later move more rapidly; (2) the different rates are properties of certain replicons that begin DNA fiber growth at different times in S; (3) the replicons of different cell subpopulations become distinctive as a result of differentiation, as shown by Callan [12]. Of these three possibilities, the first two were testable within the context of the present experiments. Therefore, SP meristems were fed 2 % sucrose for 10 h and then pulsed first for 45 min with high and then 90 min with low specific activity r3H]TdR. These meristems have cells just beginning DNA synthesis, a few in mid-S, but none in late S [16]. Upon examination of the autoradiographs of labeled DNA fibers, it became apparent that there were two distinct patterns of tandem arrays of grains. One was formed by relatively long arrays and many of these had tails of contiguous grain density gradients indicative of bidirectional fork movement (fig. 5a, d, e).
DNA fiber replication
The other pattern consisted of short tandem arrays of high grain density (fig. 5b, c, f). Fibers with one pattern or the other often were grouped on the microscope slide making selective measurements possible. One could determine both the rate of fork movement and the replicon size on the same fibers. The histograms shown in fig. 6 are data obtained from fibers with patterns of short, dense arrays of grains, such as those shown in fig. 5 b, c, $ The average length of these arrays was 7 pm and they ranged in size from 3-20 pm. However, most of the replicated segments, 78 %, were in the 5-10 pm category (fig. 6~2). The rate of DNA fiber growth was 3.1 pm h-l fork-l and the replicon size estimated from l/2 replicon lengths was 39 pm (fig. 6b). A slightly larger size was obtained from measurements of the midpoint-midpoint distances between sequential pairs of tandem arrays. These measurements had a mean of 42 pm and a mode of 35-40 pm (fig. 6~); slightly more than 22 % of the replicons were larger than 50 pm. Fibers with the longer array patterns (fig. 5a, d, e) showed grain density gradients that averaged 18 pm in length (fig. 7a). These had a mode of 10-15 pm and ranged from 3-80 pm. The average rate of fork movement was 8 pm h-l and the replicon size estimated from center-center distances was 49 pm. The shortest replicon measured was 15 pm and the longest was 125 pm. The modal replicon size was 3MO pm and approx. 40% of these were more than 50 pm long (fig. 7 b). To verify that cells in very early S have chromosomal DNA fibers with two distinctive replicon classes, midpoint-midpoint measurements were made on fibers of SP cells fed sucrose 6 h prior to a 90 min pulse with [3H]TdR. From these measurements
40
the average replicon size was estimated to be 38 pm, the modal size was 35-40 pm and 23 % were longer than 50 pm. The average rate of fiber growth was 4.5 pm h-’ fork-‘. While many of the DNA fibers had repiicons with characteristics that were consistent with those expected of smaller units with slowly moving forks, there were others that had asymmetrical arrays of varied lengths (3-55 km), i.e., there were fibers with tandem arrays of different lengths and the various arrays were not ordered in the fashion described in fig. 1b. On the fibers with less uniform arrays the average rate of movement/fork was 8.5 pm h-” and the average length of labeled DNA was 12.7 pm. The presence of these faster forks indicated that activation of replication of both classes of replicons occurred within a reiatively short time in early S. In table I are data from SP cells incubated 6 or IO sucrose. It is recognized that some of the slower moving forks only appear as such because they are of replicons that were activated during the latter portion of the pulse. However, it is unlikely that all fall into this category since the density of the grain arrays is more indicative of activity during the high specific activity pulse than during the time when the isotope was more dilute. A more attractive interpretation is that the slower forks maintain a constant pace as the cells progress through S. DISeUSSION Temporal changes in rate offork movement in S
In some plant and animal cells DNA replication is initiated by replicons in which the rate of fork movement is lower in the early portion of S than when the cells are in midor late S. For example, HeLa cells at the beginning and at the end of S have fork
402
Van’t Hof
age nuclei have replicons of approx. 3 pm movement rates of 18-30 and 66 pm h-l, respectively [17]. Also, Chinese hamster that are shorter than those of cultured cells, ovary cells in early S have forks that move but the cultured cells themselves have two 12.6 pm h-l on the average, while those ac- size groups, one of about 9 and another of tivated 7 h later move 38 pm h-l [18]. In 19 pm [21]. Also, in one of two species of another experiment with Chinese hamster Dipidomys, D. ordii, there is a group of cells the rate increased 2.6 times within a fibers with unusual replication patterns 4 h period as the cells passed from early to [22]. However, the size of these replicons is late S [18]. In both experiments the in- difficult to ascertain because of the manner creased fork rate occurred after 1 h or more. in which the measurements were made [23]. If the center-to-center distances were In pea cells the average rate of replication in very early S was about 6 while that of measured as described in ref. [23], then the sizes reported represent the distance becells that were/4,Omin into S was 18 pm h-l (fig. 4). That still higher rates of movement tween the two forks of the same replicon occur in pea in mid- or late-S is evident from rather than between two adjacent origins the fact that the average rate measured in of neighboring replicons. The two size classes of replicons in pea asynchronously dividing cells was 29 pm h-l [6]. However, precisely when in S the may be significant for three reasons, each rapid forks are activated in pea remains to of which is not exclusive of the other. (1) It is possible that the smaller replicons are be determined. The average replicon size of chromo- located on certain DNA fibers that represomal DNA fibers of pea cells at the very sent a normal part of the total genome that beginning of S and of cells that are ter- is replicated from early to late S. Assuming minating replication is 38-42 pm. This value that each fork moved at a constant rate of is 15 pm shorter than the average size meas- about 6 pm h-l, 34 h would be needed to ured from asynchronously dividing pea root replicate 38-42 pm of DNA. Thus, these cells (average size 55 pm). Approx. 20% of smaller replicons, with slowly moving the replicons of asynchronous cells are forks, are active during 60-90 % of T, and may be the major element in the determina40 pm or less and of these 15% are 35 40 pm long [6]. Also, fibers with short ar- tion of its magnitude. (2) It is likely that the smaller replicons rays that are closely spaced, as shown in fig. 5b, c, f, are found on autoradiographs reflect synthetic activity of a subpopulation of DNA of cells that never were starved or of cells within the root that are a part of treated with FUdR (see fig. 11 of ref. [6]). the growth fraction but are also differentiated anatomically within the root. If this Significance of different replicon were the case, a change in the replicon size sizes is associated with cell differentiation, as Before discussing the possible significance suggested by Callan [ 191. of replicon size classes, it should be pointed (3) The smaller replicons may have been out that cells of amphibians and insects induced by carbohydrate starvation. While have at least two size classes that are more this notion is applicable to starved cells, frequently observed. In species of Triturus it is not supported by the fact that cells never starved also have DNA fibers with a the size is different when cells differentiate [ 19,201 and in Drosophila not only do cleav- smaller replicon size class [6]. Exp CellRes 103(1976)
DNA fiber replication
As mentioned earlier, Hori & Lark [22] observed DNA fibers with replicons that had an unusual replication pattern in one of two species of Dipidomys. Species specificity, consequently, is a factor to consider when discussing replication patterns. Therefore, all inferences and interpretations pertaining to the patterns of pea DNA should be restricted to this species until further detailed data are obtained of DNA fibers of other plants. The author wishes to acknowledge the technical assistance of MS C. Bjerknes and Mr J. Clinton, both of whom along with Dr A. Kuniyuki contributed constructively to the ideas expressed in the paper. Research carried out at Brookhaven National Laboratory under the auspices of the US Energy Research and Development Administration.
REFERENCES 1. Kovacs, C J & Van? Hof, J, Radiat res 48 (1971) 95. 2. Van’t Hof, J, Advances in radiation research (ed J F Duplan & A Chapiro) vol. 2, p. 881. Gordon & Breach Science Publ., New York (1973).
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Van’t Hof, J, Radiat res 41 (1970) 538. - Exp cell res 37 (1965) 292. -J cell bio127 (1965) 179. - Exp cell res 93 (1975) 95. -Ibid 99 (1976) 47. Webster, P L & Van’t Hof, J, Am j bot 57 (1970) 130. 9. Conger, A D & Fairchild, L M, Stain technol 28 (1953) 281. 10. Huberman, J A & Tsai. A J, 5 mol biol 75 (1973) 5. 11. Huberman, J A & Riggs, A D, J mol biol32 (1968) 327. 12. Callan, H G, Brit med bull 29 (1973) 192. 13. Van’t Hof, J, Brookhaven symp bio125 (1973) 152. 14. Kovacs, C J & Van? Hof, J, J cell biol 47 (1970) 536. 15. Ockev, C H, EXD cell res 70 (1972) 203. 16. Van’tHof, J, J cell bio137 (1968) 773. 17. Painter, R B & Schaefer, A W, J mol biol 58 (1971) 289. 18. Housman. D & Huberman. J A. J mol biol. 94 (1975) 173. 19. Callan. H G. Cold Sorine Harbor svmo I L.want bioi 38 (1974) 195, - 20. Tavlor. J H. Int rev cvtol37 (1974) 1. 21. Blumenthal; A B, Kridgstein,‘H J h Hogness, D S. Cold Spring Harbor symp quant bio138 (1974) 205. 22. Hori, T & Lark, K G, J mol biol88 (1974) 221. 23. - Ibid 77 ( 1973)391. 3. 4. 5. 6. 7. 8.
Received June 2 1, 1976 Accepted July 7, 1976