Experimental

DNA

FIBER

Ceil Research 93 (1975) 95-104

REPLICATION

OF A HIGHER

PLANT

IN CHROMOSOMES (PZSUM

SATZVUM)

J. VAN’T HOF Biology Department, Brookhaven National Upton, NY 11973. USA

Laboratory,

SUMMARY Techniques were developed for the autoradiographic analysis of chromosomal DNA fiber replication of higher plant cells. The DNA fibers of Pisum root meristem cells replicate via many tandemly arranged replicons. Each replicon has an initiation point and the average distance between them is about 54 Wm. Most of the replicons of Pisum showed bidirectional DNA chain growth that proceeded from a commonly shared initiation point. The average rate of replication per single fork is approx. 29 Fm/h at 23°C.

The autoradiographic analysis of chromosomal DNA chain growth has been performed on bacteria [l-3], T4 phage [4], mammalian [5-71, amphibian [8], and avian cells [9]. Cells of all higher organisms studied to date have long chromosomal DNA fibers that are replicated in tandemly joined sections each of which has an initiation point. In 1963 Taylor [lo] postulated that eukaryotic chromosomes were composed of “tandem-linked series of ‘replicons’ “, which were “segments of DNA which replicate as units . . .“. DNA chain growth in cells of higher organisms is accomplished by two replicating forks that travel in opposite directions from an initiation point within each replicon. The distance between initiation points of mammalian chromosomal DNA is approx. 60 pm in Chinese hamster cells [7] and as near as 17 pm in MDBK bovine cells [l 11.The rate of replication per single fork is between 30 and 72 7-751809

pm/h with the average more near the lower of the two values. The cells of the amphibians, Triturus and Xenopus, have chromosomal DNA fibers with initiation points that on the average are 60 pm apart in the latter and range from 100 to 350 pm apart in the former species [8]. The rate of replication per single fork in Xenopus is 9 pm/h and that of Triturus is 20 pm/h [8]. Avian cells (Gallus domesticus) in culture, have statistics similar to those of Chinese hamster. The initiation points are separated by 63 pm and they have a rate of replication per single fork of 24 to 30 pm [9]. Among the factors that determine the duration of the cell cycle in unrelated plant species is the amount of nuclear DNA [12141. It is equally clear that the control of cell division in higher plants involves both the initiation of DNA synthesis and the onset of mitosis [IS]. Even though the amount of nuclear DNA and the initiation of DNA synthesis are known to be of importance Exprt Cell Res 93 (1975)

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to plant cell division there are no data on chromosome DNA chain growth comparable to those of mammalian, avian, and amphibian cells. To understand why the cell cycle duration increases with nuclear DNA content in unrelated plant species it is necessary to determine the number of replicons per cell and the rate at which the DNA molecule is replicated. These two factors may well contribute to the overall duration of S. The experiments to be described in this paper represent the first with plant cells and they characterize DNA chain growth in the chromosomes ofPisum.

mixture, single-drop quanttttes ol the tollowtng \o111tions were added in sequence: SSC. pH 6.8 (NaCI, 0.14 M; Na citrate, 0.015 M; NaH?PO,. 0.5 MI. 2xlO~?I EDT.4, and 5% sodium laural sulfate dissolved in 0.1) M Na-citrate and 0.05 M Tris buffer. pH 7.4. The mixture of liqurd was gently spread over the surface of the slide by tilting it and then the material wa\ oven dried at 37°C. The slides were then treated according to the protocol of Hubberman & Tsai [ 191which consisted of two successive 5 min rinses in ice-cold 5% TCA and one 3 min rinse in Y5% ethanol. When dry. the slides were dipned in Kodak NTB liquid emulsion dried again, and.exposed for 30 days -at 4°C in the presence of a desiccant. The autoradiographs were developed in Kodak D-19 at 22°C for 12 min. tixed. washed, dried, and mounted under the coverglass with Euporal. The autoradiographs were examined and measured by bright field microscopy.

RESULTS AND DISCUSSION MATERIALS

AND METHODS

The primary root tip meristem of cultured Pisurn roots was the source of cells in the present study. The general techniques for Pisum root culture were published previously [l6]. In the present experiments only White’s medium with 2 % sucrose was used. After 4 to 5 days in culture the terminal 1.5 to 2.0 mm tip was cut and suspended in 0.25 ml aqueous sterile isotope solution at 23°C. The high radioactivity pulses were performed with aqueous tritiated thymidine (3H-TdR) at 1000 &i/ml which had a specific activity of 50.6 Ci/mM (New England Nuclear, Boston, Mass.). The low radioactivity pulses were carried out with “H-TdR having a specific activity of 6 Ci/mM and at a concentration of 1000 &i/ml. The duration of the isotope pulses varied with experiment and are appropriately noted in the text. After a given pulse or a series of pulses the root tips were sispended for 5 min in icecold buffered 2% formaldehvde [I71 and immersed in 0.132 M phosphate buffer (pH 6.8j until the nuclei were isolated Isolation and lysis of nuclei were performed with single root ti s using modifications of the methods of McLeish [I7 f and Lark et al. [18]. Each root tip was placed between two dry microscope slides previously coated with 0.5 % gelatin, 0.05 % CrK(S04*. 12 H,O, a drop of 0.132 M phosphate buffer was added and the tissue was squashed by pinching the slides together very tightly. The drop of liquid that formed at the edge of the two slides contained a suspension of nuclei and this drop was placed on another coated dry slide. To the nuclear susoension was added a dron of trvpsin (0.5 mg/ml of 0.>32 M phosphate buffer, pH 6.8): -The enzvme was allowed to react with the nuclei for about 15 min or until lysis occurred. To the trypsin-nuclear

The direction of chain growth of chromosomal DNA fibers To determine whether DNA chain growth was bi- or unidirectional a ‘step-down’ experiment was performed. The labeling protocol consisted of a 45 min pulse with high specific activity “H-TdR followed immediately by a 30 min pulse with low specific activity 3H-TdR. The difference in specific activity was &fold and sufficient to detect the direction of replication [19]. Autoradiograms of tandem arrays of labeled chromosomal DNA fibers produced by the ‘step-down’ protocol are shown in figs 1.4. In fig. 1 are tandem arrays of silver grains on an autoradiograph that reflect the manner of DNA chain growth in adjoining replicons. Contiguous grain density shifts are visible in some of the five autoradiograms. The first autoradiogram that is located in the upper right hand corner of the photograph has a high density of grains that is not bounded on either side by tails of less dense contiguous grain arrays. The

Figs 1-I. Photographs of chromosomal DNA fiber autoradiographs from meristematic cells of Pisunr root tips cultured at 23°C. The tissue was first pulsed 45 min with high specific activity “H-TdR (50.6 Ci/mM) and then pulsed for 30 min with low specific activity 3H-TdR (6 Ci/mM). Fig. 4 is a higher magnification of fig. 3. The dark line scale is 50 pm long. Exptl Cell Res 93 (1975)

DNA fiber replication

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J. Van’t Huf

second autoradiogram downward. how- single fork was about 30 pm/h. I‘he pho!oever, has a grain pattern that is unmistak- graph in this case is of sufficient clarity to ably characteristic of bidirectional chain show the two sister strands of the DNA growth, i.e. DNA chain growth that be- fiber in the tail that is proceeding from left gins at a given point and proceeds from that to right. Continuing downward on the point in two opposite directions. The cen- photograph, toward the lower right hand tral portion is more dense than either side side, are two closely associated arrays each and the grain density shifts are contiguous. with grain density gradients going in one The dense central portion was replicated direction from left to right. Assuming that during the period of the high radioactivity the more dense portions were replicated pulse and the less dense tails were formed during the high radioactivity pulse and the during the period of the lower radioactivity less dense tails when the radioactivity was pulse. Continuing downward on the photo- less, these autoradiograms reflect unidirecgraph we see the third and fourth grain ar- tional chain growth. rays. These are short (about 13 pm long) The combination of adjacent replicons, and close to each other. Taken together one which replicates bidirectionally and they suggest an autoradiographic pattern another that possibly increases chain expected from bidirectional chain growth growth unidirectionally, is shown in figs 3 that was initiated before the high radioac- and 4. Fig. 3 offers an overall view of the tivity pulse but continued during it. Thus area in which the tandem arrays are lothe 7 to 8 ym between them was replicated cated and fig. 4 is a higher magnification before the isotope was available. The last of some of the more interesting autoradioarray of grains in the photograph near the lower left hand corner again shows a pat- Table 1. Center to center distance (q) chain between tandem autoradiograms produced tern indicative of bidirectional growth. The highly dense central portion by a ‘step-down’ labeling protocol of chrowas replicated during the high radioac- mosomal DNA fibers isolated from Pisum tivity pulse and the lesser dense tails were root tip meristem cells synthesized during the period of lower raDistance dioactivity pulse. Number 92 (i-m) The autoradiograms in fig. 2 show a pat20-25 1 0.9 tern of grain densities indicative of bidirec25-30 I 0.9 tional replication in one instance and pos30-35 5 4.4 3540 17 15.2 sibly unidirectional growth in another. In 40-15 5 4.4 the left hand portion of the photograph is 45-50 I5 13.4 50-55 I8 16.1 an array approx. 78 pm long which has a 55-60 16 14.3 dense central portion and two contiguous 60-65 I5 13.4 tails of a lesser density. Since the com65-70 10 8.9 70-75 2 1.8 bined duration of the two 3H-TdR pulses 75-80 2 1.8 80-85 I .8 was 75 min the rate at which chain growth 90-95 : 1.8 was occurring in this particular replicon I 0.9 105-I 10 I 0.9 110-115 was a little more than 60 pm/h. Further, I 0.9 135-140 because the growth was bidirectional two forks were involved and thus the rate per Number measured, 112; average, 54.67 Frn. Exstl Ceil Res 93 (1975)

DNAfiber replication

99

grams shown in fig. 3. The autoradiogram near the center of fig. 4 has a pattern of grain density gradients that run in opposite directions from the more highly dense central area. DNA chain growth in this replicon is bidirectional. The adjoining arrays above and below it however, have only one tail of decreasing grain density and may reflect unidirectional chain growth or even asynchrony in the initiation of replication between the two forks of a given replicon. The distance between initiation points Callan mentioned in 1972 [8] that the distance between initiation points of adjacent replicons can be determined from a ‘stepdown’ pulse experiments by measuring the center-to-center spaces between tandem arrays of grains for which the direction of replication was unambiguous. Following this guideline the distances tabulated in table 1 were obtained. The average distance between initiation points was 54.7 km and they ranged from 20 to 140 brn. Of the 112 autoradiograms measured and scored, over 90% showed grain density patterns indicative of bidirectional replication. Rate of chromosomal DNA chain growth Having established that most of the tandem replicons on the DNA fiber replicate bidirectionally, the rate of DNA chain growth was determined by a series of single pulse experiments with high radioactivity 3HTdR. Pulses of 15, 30, and 90 min were used and the rate of replication was derived from the measurement of the length of the tandem arrays of silver grains. The histograms in fig. 5 show the distribution of lengths of the arrays produced by the three different pulses. After a 15 min pulse the length ranged from 2 to 22 pm and averaged about 7.4 pm long. In hourly units this average indicates a rate of 29.6 pm/h/

length (,um); ordinate: frequency. Histograms of the length of labeled DNA segments in tandem arrays in fiber autoradiographs after cultured Pisum root tips were pulsed for either 15, 30, or 90 min with high specific activity 3H-TdR (50.6 Ci/mM). N, Number of labeled segments measured.

Fig. 5. Abscissa:

single fork. Examples of tandem arrays of grain densities produced after a 15 min pulse are shown in figs 6 and 7. The incomplete loop in fig. 6 is a little less than 500 pm long and the fiber shown in fig. 7 is much shorter. In each instance, however, the arrays are very short compared to those produced by a 30 min pulse (fig. 8). The autoradiograms after a 30 min pulse had an average length of 14.3 pm and the sizes ranged from 2.5 to 40 pm (fig. 5). In hourly units the 30 min pulse indicated a rate of 28.6 pm/h/single fork. A 90 min pulse produced autoradiograms of labeled DNA that ranged in length from 2 to 245 pm and the average length was 21.9 ,um (fig. 5). The rate calculated from the 90 min pulse data was 14.6 pm/h/single fork. The longer length of labeled DNA (fig. 9) and the lower hourly rate calculated after a 90 min pulse are significant in two ways. (1) The rate should be less than that estimated after shorter pulses, if initiation points are separated by an average distance of 54 pm

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J. Van’t Hqf

and single forks move at a rate of nearly 30 pm/h. Given bidirectional replication tandem replicons should join within 1 h, and 90 min is in excess of this time. (2) Very long sections of labeled DNA fiber should be evident after a 90 min pulse as they represent tandem replicons that completed replication during the pulse (fig. 5). In an indirect way the lower rate calculated from the length of autoradiograms after a 90 min pulse substantiate an interval of 54 pm between initiation points and single fork rate of nearly 30 pm/h. Ver$kation of an average of 54 pm between initiation points Among the autoradiograms produced by a 90 min or a ‘step-down’ pulse were many exceptionally long DNA fibers (up to 2800 pm) with many tandem arrays of grains. The number of arrays on these long fibers was counted and expressed as a function of fiber length. The plot forms a linear curve (fig. 10) that is described by the regression equation y = -0.222 +0.03 I x with a correlation coefficient of 0.994. The fact that the curve is linear over a length of up to 2800 pm per fiber indicates that the number of replicons per fiber is independent of its total length. Also the reciprocal of the slope provides a check of the distance between initiation points. Because most replicons have two forks per single initiation point, the reciprocal must be divided by one-half. However, the quotient should be an overestimate of the distance between initiation points because, first, some of the arrays will

represent two forks from adjoining replicons that completed replication and met during the pulse. These will he scored as one array even though they represent two forks. Secondly, some of the replicons will commence replication during the pulse and the two diverging forks will again be represented by a single array. The reciprocal of the slope for the curve in fig. 10 is 32.2 and divided by one-half provides an estimate of the distance between initiation points of 64.4 pm. This value corresponds to an overestimate of 18% when compared with * that obtained from direct autoradiogram measurements from the ‘step-down’ experiment. It is important that 64 pm is an overestimate, since an overestimate adds credence to the direct measurement average of 54 pm. Diversification of chromosomal DNA chain growth Early work by Taylor [20] with Crepis capiflaris and by Wimber [21] with Tradescantia paludosa showed that chromosomes do not duplicate synchronously in the S period. After a pulse of 3H-TdR it was noted that the isotope was not incorporated evenly along the axis of the chromatids and some chromosomes did not incorporate the tracer at all. Experiments with plant [22] and animal cells [23, 241 further demonstrated that the rate of 3H-TdR incorporation during S varied and suggested that possibly DNA synthesis occurred in spurts or pulses during S. Also the autoradiographic analyses of eu- and

Figs 6-7. Photographs of autoradiographs of tandemly labeled segments ofPisum

DNA fibers after the tissue was pulsed for 15 min with high specific activity 3H-TdR. The scale is 50 @rnlong. Fig. 8. Photograph of autoradiographs of tandemly labeled segments of Pisum DNA fibers after the tissue was pulsed for 30 min with high specific activity 3H-TdR. The scale is 50 pm long. Fig. 9. Photograph of an autoradiograph of tandemly labeled segments of Pisum DNA fibers after the tissue was pulsed for 90 min with high specific activity 3H-TdR. The scale is 50 @rnlong. Exprl Cell Res 93 (1975)

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heterochromatin synthesis tn both plant [2S, 261 and animal cells [25] showed that the different chromatins are replicated at different times in S. The cells of the root tip meristem ot Pisum are not different from those of Crcpis or Tradrscantiu; they also replicate chromosomes asynchronously in S (Van? Hof, unpublished observations). It is recognized that in an asynchronous cell population the replication of chromosomal Fig. 10. Abscissa: length (pm); ordinate: no. of arrays. DNA fibers will reflect the asynchrony. The number of tandem arrays of grains expressed as a function of fiber length measured on autoradiograms For example, the autoradiograms shown in (_ of labeled Pisunt DNA after the tissue was either figs I1 and 12 and those in fig. 9 were pulsed for 90 min with high specific activity 3H-TdR or given a ‘step-down’ pulse. Corr. coef. is the correla- produced by labeled DNA fibers that were tion coefficient of the regression line the parameters of pulsed for 90 min. The arrays of silver which are shown in the figure. The regression line is discontinuous because of a change in the increments grains in fig. 11 are short (about 6 pm) and on the abscissa after 800 pm. close together. (Assuming two forks and one initiation point per replicon, the distance between initiation points is about 20 pm.) The shortness of the tandem ar#/

50’

Figs 11-12. Photographs of autoradiographs of tandemly labeled segments of Pisum DNA fibers after the tissue was pulsed for 90 min with high specific activity 3H-TdR. The dark line XI de is 50 pm long. Exprl Cell Res 93 (1975)

DNA fiber replication

rays represents a peculiarity of this particular DNA fiber in that either the rate of chain growth was much lower than that of other fibers in the genome or replication did not begin until the 90 min pulse was about to be terminated. Whatever the cause of the uniformity of size of the arrays on this particular DNA fiber, it indicates that within a given genome there are groups of replicons on certain DNA fibers that initiate replication synchronously and increase chain growth at nearly the same rate. A similar observation was made by Hand & Tamm [l l] recently. They state that in mammalian cells, “initiation events occur in bursts and there appears to be a degree of synchrony among individual events”. The fact that several replicons along the same DNA fiber act in unison suggests that they may be responding to a common trigger or stimulus. The lack of uniformity, however, is common as shown in fig. 12. Here the labeled DNA segments are longer than those shown in fig. 11 and shorter than those displayed in fig. 9. The general pattern of the autoradiograms in fig. 12 strongly suggests sister strand separation and the presence of two forks, one pointing toward the top and the other toward the bottom of the photograph. The sister strands are separated over a distance of more than 60 pm and each has a similar pattern of tandem arrays of grains that lie nearly side by side. In the root tip meristem ofPisum the cells in G 1 have a 2C DNA amount of approx. 7.9X1O-12 g [14]. Before these cells reach mitosis an equal amount of DNA must be replicated. The 2C amount of DNA corresponds to about 240 cm in length and 17 cm per chromatid (2n= 14). With the assumption that (1) the average distance between initiation points is about 54 vrn; and (2) each chromatid consists of a single

103

long DNA fiber, the number of initiation points per chromatid is estimated to be approx. 3 100. Therefore a cell in G 1 has about 43000 initiation points or replicons that function at one time or another as it proceeds through S and into G 2. The number of replicons estimated for Pisum agrees well with that determined from Xenopus by Callan [8]. Callan’s value for a haploid (1C) nuclear DNA amount was 3 x lo-l2 g and the number of replicons in such cells ranged from 15 to 16000. A somatic cell in G 1 has a 2C nuclear DNA value and the amount of DNA is 6x lo-l2 g; thus, the estimated number of replicons likewise is doubled to 30 to 32000. A simple comparison of the data from Pisum with those of Xenopus shows that the number of replicons is roughly proportional to the amount of nuclear DNA as suggested by Callan. However, while the relation holds well for Pisum and Xenopus, it fails to apply strongly in the case of Triturus [8]. The author wishes to thank W. Geisbusch for assistance with the photography. This research was carried out at Brookhaven National Laboratory under the auspices of the US Atomic Energy Commission.

REFERENCES 1. Cairns, J, J mol biol6 (1963) 208. 2. Dennis,ES&Wake,RG, Jmolbiol15(1966)435. 3. - Replication and recombination of genetic material (ed W J Peacock & R D Brock) p. 61. Australian Acad Sci, Canberra (1968). 4. Cairns, J, J mol bio13 (1961) 756. 5. - Ibid 15 (1966) 372. 6. Hubberman, J A & Riggs, A D, Proc natl acad sci US 55 (1966) 599. 7. - J mol biol32 (1968) 327. 8. Callan, H G, Proc roy sot LondonB 181(1972) 19. 9. McFarlane, P W & Callan, H G, J cell sci 13 (1973) 821. 10. Taylor, J H, J cell physio162 suppl. 1 (1963) 73. Il. Hand, R & Tamm, I, Cell cycle controls (ed G M Padilla, I L Cameron & A-Zimmerman) p. 273. Academic Press, New York (1974). 12. Van? Hof, J & Sparrow, A H, Proc natl acad sci US 49 (1963) 897. 13. Van? Hof, J, Exptl cell res 39 (1965) 48. Exptl Cell Res 93 (1975)

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14. Survey of genetic\ ted K King) vol. 7. p. 363. Plenum Publ. Co.. New York (lY74). 15. Van’t Hof, J. Cell cycle controls ted P M Padilla. I L Cameron & A Zimmerman) D. 77. Academic Press, New York (1974). 16. Van’t Hof, J. Am j bot 53 (1966) Y70. 17. McLeish, J, Proc roy socB London IS8 (1963) 261. 18. Lark, K G, Consigli. R & Toliver, A, J mol biol58 (1971) 873. 19. Hubberman, J A & Tsai, A, J mol biol75 (1973) 5. 20. Taylor, J H, Exptl cell res I5 (19.58) 350. 21. Wimber. D E, Exptl cell rer 23 (1961) 402.

Exptl Cell Res 93 (I 975 )

22. Howard, .4 & Dewey. 11 I.. I-.:upti CCII ~-es 14 (IY61 I 623. 23. Klevecz. R R & Kapp. 1. N. J cell hlol 5X tic)731 564. 24. Remington. J .A 6i Klevecr. K R. i..xptl cell r :\ 76 (1973) 410. 25. Lima-de-Faria, A, Handbook of. molecular cytology (ed A Lima-de-Faria) p. 277. North-Holland, Amsterdam (1969). 26. Masubuchi, M, Bot mag Tokyo 86 t 1973) 3 19. Received

November

26. IY74

DNA fiber replication in chromosomes of a higher plant (Pisum sativum).

Experimental DNA FIBER Ceil Research 93 (1975) 95-104 REPLICATION OF A HIGHER PLANT IN CHROMOSOMES (PZSUM SATZVUM) J. VAN’T HOF Biology Depar...
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