Plant Molecular Biology 20: 377-382, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
377
Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis Jack Van 't Hof and Susan S. Lamm
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA Received 18 November 1991; accepted in revised form 14 May 1992
Key words: replication, origin, rDNA, 5-aminouracil, Pisum sativum, synchrony Abstract
The time course of replication of the 9 kb ribosomal D N A repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.
Introduction
Previously we located the replication origin of the 9 kb ribosomal D N A repeats (rDNA) of pea in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene [9]. The method used to localize the origin, however, did not provide the time-dependent evidence expected of replication initiation followed by fork movement within the replicon. Here we show that indeed replication starts in the r D N A fragment in which we found the origin to be located, and then proceeds to the adjoining r D N A sequences within the repeat. This process is detectable using less than 0.5 #g ofgenomic D N A from root meristems in which 6 ~o or less of the cells are replicating their DNA. Materials and methods
[ 18, 19]. Meristematic cells of primary roots were synchronized by suspending roots of seedlings for 12 h at 20 °C in an aerated solution containing nutrient salts and 5-aminouracil (0.2mg/ml; Sigma A-4005). 5-aminouracil temporarily blocks dividing cells in late S phase. The blockage is removed when seedlings are transferred to fresh nutrient salt solution containing no inhibitor. After treatment meristematic cells recover and divide synchronously within 7 h. To determine when and how many cells divided, 2 to 3 mm tips of primary roots were fixed with 3 parts methanol, 1 part glacial acetic acid, hydrolyzed with 5N HC1 at 22 °C for 30 to 45 min, stained either by the Feulgen method or with toluidine blue, and squashed on microscope slides. Dividing cells were counted from random samples on the slides, 1000 cells per meristem, three meristems per sample.
Ceil synchronization
Single-cell autoradiography
Pea seeds (Pisum sativum cv. Alaska) were germinated and grown as previously described
Cells in S phase at the 6th h of recovery were labeled for 1 h with 3H-thymidine (3 #Ci/ml; sp.
378 act. 55 Ci/mmol) by immersing the primary roots of seedlings in 250 ml of nutrient solution containing the isotope. After transfer to nonradioactive medium, root tips were fixed at hourly intervals up to 13 h, stained by the Feulgen method, squashed on microscope slides and processed for autoradiography by published procedures [16, 17]. The autoradiograms were scored for percent labeled division figures to determine when and how many of the radioactive cells divided. All other methods used in these experiments are as described by Van't Hof and Lamm [19] unless noted otherwise in the figure legends.
Results and discussion
Design of the experiment At least three factors must be considered in designing an experiment with a high probability of detecting replication initiation in pea root cells. One factor is cytological. Since r D N A is replicated throughout S phase [20], r D N A from asynchronously dividing cells will yield replicons in all stages of replication: initiation, chain elongation and termination. To enrich for replicons in the initiation stage, we used an inhibitor, 5-aminouracil, to block cells in late S phase. Upon removal of the blockage, we anticipated that cells still in S phase would initiate replication of their genome and complete the process while other cells, such as those of the quiescent center, would begin D N A replication in response to the effects of 5aminouracil [4, 5]. Given a situation where cells can be manipulated so they begin replication synchronously, a second factor must be considered. This factor is a means of distinguishing between replicative intermediates and non-replicating molecules. In the present experiments we used twodimensional gel electrophoresis because this method distinguishes between replicative and non-replicative molecules and also separates them [ 1, 2, 3, 12, 19]. The final factor to consider is the probes used to identify which sequence within the r D N A repeat functions as a replication origin. The probes chosen should hybridize with
specific sequences located at different positions within the repeat.
Division of synchronized cells Root meristem cells when treated with 5aminouracil continue D N A synthesis at a reduced rate [16] and are blocked in late S phase of the cell cycle [8, 13, 14, 15, 16]. The blockage is temporary, however, as cells divide eventually even in the presence of the inhibitor [13, 21, J. Van't Hof, unpublished results]. When in place, the block prevents cells from proceeding from S to G2 phase. Thus, during the first 4 h of treatment, cells continue to divide in decreasing numbers and after 12 h of treatment meristems have few, if any, dividing cells. Recovery commences when the inhibitor is removed. At the 7th hour of recovery cells divide reaching 35~o of the total number of cells in the root tip by the 8th hour (Fig. 1). Cell division remains near this level for 1.5 h (9.5 h in Fig. 1) and then decreases to control level at 12 h.
50 45 _~
40
o
35
"0
25 "O
.
20
c
o
15
a.
10~
untreated control
3 ,I'
0
~' Hours
~ after
H " ",,,,,,,
1'o
¢1
1'~
1~
5-AU t r e a t m e n t
Fig. 1. The percent dividing cells in the meristematic tip of primary roots of pea seedlings expressed as a function of time after a 12 h treatment with 0.2 mg/ml 5-aminouracil. Open triangles, non-radioactive dividing cells; dotted line, radioactive dividing cells labeled with 3H-thymidine for 1 h 6 hours after 5-aminouracil treatment; dashed line (control), dividing cells in root tips neither treated with 5-aminouracil nor labeled with isotope.
379 The 1.5 h span in which the number of dividing cells remains relatively constant is roughly equivalent to the duration of mitosis in pea [17]. Thus, cells in prophase at 8 h will be in anaphase and telophase at 9.5 h. As this cohort completes division the number of dividing cells decreases. Following this first cohort, a smaller one begins to divide at 9.5 h and it too, completes division in about 1.5 h. Among the smaller cohort are cells that incorporate ~H-thymidine at the 6th hour of recovery (Fig. 1). These cells represent 6~o or less of the total number of dividing cells in the meristem. We do not know the source of the cells in our experiment but the work of Clowes indicates that they very likely originate from the quiescent center [4, 51.
work with electron microscopy show 83, 92 and 94~o of the molecules in spots A, B and C, respectively, to be linear. The probes, 790, 1.3 and 1.6, are homologous to r D N A sequences located at increasing distances from the origin. Probe 790 hybridizes to the replication origin and two contiguous subrepeats upstream from the origin, probe 1.3 to 1.3 kb located ca. 1 kb to the right of the origin and contains ARS consensus sequences [9] and probe 1.6 to coding sequences located about 3 kb downstream to the right of the origin. Since the r D N A repeats are tandem, probe 1.6 also hybridizes to sequences about 4 kb upstream to the left of the origin.
Spots A, B and C are transitory Detection of single-stranded replication fragments of rDNA Ribosomal D N A of pea, either digested with Eco RI or double-digested with Eco RI and Hind lII and separated by neutral-neutral twodimensional electrophoresis, produces three spots of single-stranded replicative intermediates [ 19]. In the second dimension these spots migrate as an arc above another, dominant, arc of duplex molecules. The r D N A sequences in the spots, the length of the single-stranded chains they contain and the probes to which they hybridize are given in Table 1. More recent data from preliminary Tablel. Summary of molecular characteristics of spots A, B, and C containing single-stranded replication intermediates of r D N A of pea
Spot
Size (kb)*
Hybridization to probes
rDNA sequences
790
1.3
1.6
A
9
+
+
+
B
5
+
-
+
C
3.6
+
+
-
spacer, 18S, 5.8S, 25S spacer, 25S spacer, 18S
Data from Van't Hof and Lamm [19]. * Determined by alkaline gel electrophoresis.
When replication is reduced or stopped the spots are either absent or only slightly visible when hybridized to specific probes. When replication is resumed, the spot containing the replication origin should appear first. Later on, after replication is beyond the initiation stage, all three spots should be detectable. Evidence that these changes occur is shown in Fig. 2. The autoradiograms are of fragments of r D N A produced by double digestion with Eco RI and Hind III. Each blot was probed with 790, a sequence that hybridizes to all three spots [19]. The two arcs in the autoradiograms reflect the different migration rates of replicative and nonreplicative r D N A fragments in the seconddimension gel. The darker lower arc contains duplex r D N A fragments that are not replicating [19]. The upper arc has single-stranded replication intermediates seen as three spots, labeled A, B and C. Note that each autoradiogram has spots but their size and hybridization signal differ with the source of rDNA. The signals are weak if the r D N A is from cells given 3 h to recover (Fig. 2a) strong, if the r D N A is either from cells given 12 h of recovery (Fig. 2b) or from untreated (control) asynchronously dividing cells (Fig. 2c). We interpret these results to indicate that little r D N A replication occurs 3 h after 5-aminouracil treatment
380
Fig. 2. Identification of replication intermediates of ribosomal D N A of pea root cells. Autoradiograms of Southern blots of ribosomal D N A double-digested with Eco RI and Hind III. Fragments were separated by two-dimensional gel electrophoresis run under neutral conditions in both directions and probed with 790. a) r D N A fragments from meristematic cells 3 h after being treated for 12 h with 0.2 mg/ml 5-aminouracil; b) fragments from cells 12 h after treatment; c) fragments from untreated (control) meristematic cells. A, B and C are spots of replicative intermediates on an arc located above a darker arc formed by linear duplex r D N A fragments. The numbers, in kb, refer to duplex rDNA fragments. Direction of 1st dimension (0.5% agarose; 0.66 V/cm, 19.5 h), right to left; 2nd dimension, (1.75% agarose, a; 1.5% agarose, b and c; 10 V/cm, 2.5 h), top to bottom.
Fig. 3. Autoradiograms of a Southern blot of ribosomal D N A Eco RI-Hind III fragments from pea root meristems separated by two-dimensional gel electrophoresis under neutral conditions in both directions. The blots were probed, stripped and probed again sequentially with 790, 1.3 and 1.6. The probes are noted above the autoradiograms, a), b) and c) fragments from cells 6 h after treatment with 5-aminouracil; d), e) and f) fragments from cells 8 h after treatment. Spots A, B and C, numbers and direction of migration are as described in Fig. 2; 1st dimension 0.5% agarose, 0.66 V/cm, 19.5 h; 2rid dimension 1.75% agarose, 10 V/cm, 2.5 h.
and that it is resumed by the 12th hour. Replication began, therefore, some time between the 3rd and 12th h after treatment.
Evidence that r D N A replication begins 6 h after treatment is shown in Fig. 3, the top row of panels. The autoradiograms in these panels are of
381 the same blot of r D N A probed, stripped and probed again sequentially with the three probes noted above the panels. Note that probe 790 hybridized strongly only to spot C (Fig. 3a). Probe 1.3, likewise, hybridized only to spot C but the signal is weak (Fig. 3b). On the other hand, probe 1.6 failed to hybridize to any spot (Fig. 3c). It is significant that probe 790 gave a strong signal with spot C and weak signals with spots A and B. This probe hybridizes to all spots, if they are present on the blot (Figs. 2b and 2c). The weak hybridization to spots A and B, therefore, indicates that sequences contained in these spots are not replicating at 6 h. If replication begins in sequences contained in spot C, then as time passes active replication forks are expected to move to sequences contained in spots A and B. As seen in Fig. 3, the bottom row of panels, an additional 2 h of recovery does produce the expected result. Autoradiograms of blots of ribosomal D N A from cells 8 h after treatment show three spots when probed with 790 (Fig. 3d), two (A and C) when probed with 1.3 (Fig. 3e) and two (A and B) when probed with 1.6 (Fig. 3f). Taken together, the autoradiograms in Fig. 3 provide evidence that the replication of the r D N A repeats follows a temporal order and that replication begins in sequences contained in spot C. The sequences in spot C are those of the spacer region including the subrepeats. Among the subrepeats, approximately 1.5 kb downstream from the 3' end of the 25S gene, is a 209 base sequence identified previously as the replication origin [9]. Finally, it should not be overlooked that the results presented here were obtained using less than 0.5 #g of genomic D N A per gel. Of this DNA, approximately 0.08~o or 0.4 ng is rDNA. This is enough r D N A to detect replication intermediates, given that there are about 3900 copies of ribosomal genes in pea var. Alaska [7, 10]. Previously, we demonstrated that the replication intermediates are single-stranded and products of serial replication displacement loops [ 19]. Since these intermediates are detectable with as little as 0.5/~g of genomic DNA, replication by serial displacement loops is likely the most common, but
not necessarily the only, mechanism of r D N A replication in pea. The meristem is a complex tissue composed of several distinctive cell populations [6] a feature that distinguishes it from single cell systems such as yeast and cultured mammalian cells. The response of cells in the meristem to 5-aminouracil treatment, therefore, may differ from that expected of single cell systems. For example, in the meristem, mitotic inhibition of most cells is the signal for others in the quiescent center to initiate D N A replication and divide [4, 5]. Further, meristematic cells may differ from yeast cells in the means used to replicate rDNA. Yeast cells replicate r D N A unidirectionally via leading and lagging strands [3, 12] while in pea the data support replication by serial displacement loops [ 19]. On the other hand, in view of the variety of mechanisms used by viruses to replicate their genome [ 11 ] it is possible that higher plants use more than one mechanism to replicate their genome.
Acknowledgements Research was supported by the Office of Health and Environmental Research, US Department of Energy.
References 1. Bell L, Byers B: General method for separating branched DNA molecules. Anal Biochem 130:527-535 (1983). 2. Brewer BJ, Fangman WL: The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51: 463471 (1987). 3. Brewer BJ, Fangman WL: A replication fork barrier at the 3' end of yeast ribosomal RNA genes. Cell 55: 637643 (1988). 4. Clowes FAL: X-irradiation of root meristems. Ann Bot 27:343-352 (1963). 5. Clowes FAL: Synchronization in a meristem by 5-aminouracil. J Exp Bot 16:581-586 (1965). 6. Clowes FAL: The control of cell proliferation within root meristems. In: Miller MW, Kuehnert CC (eds) The Dynamics of Meristem Cell Populations, pp. 133-147. Plenum Press, New York (1972). 7. Cullis CA, Davies DR: Ribosomal D N A amounts in Pisum sativum. Genetics 81:485-492 (1975). 8. Diez JL, Gonzfilez-Fernfindez A, L6pez-Sfiez JF: Mech-
382
9.
10. 11. 12.
13.
14.
15.
anism of mitotic synchronization induced by 5-aminouracil. Exp Cell Res 98:79-89 (1976). Hernfindez P, Bjerknes CA, Lamm SS, Van't Hof J: Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum). Plant Mol Biol 10:413-422 (1988). Ingle J, Sinclair J: Ribosomal RNA genes and plant development. Nature 235:30-32 (1972). Kornberg A: D N A Replication. WH Freeman & Co, San Francisco (1980). Linskens MHK, Huberman JA: Organization of replication ribosomal D N A in Saccharomyces cerevisiae. Mol Cell Biol 8:4927-4935 (1988). PrenskyW, Smith HH:Themechanismof5-aminouracilinduced synchrony of cell division in Viciafaba root meristems. J Cell Biol 24:401-414 (1965). Scheuermann W, Klaffke-Lobsien G: On the influence of 5-amino uracil on the cell cycle of root tip meristems. Exp Cell Res 76:428-436 (1973). Socher SH, Davidson D: 5-aminouracil treatment: a method for estimating G2. J Cell Bio148:248-252 (1971).
16. Van't H o f J : Experimental control of D N A synthesizing and dividing cells in excised root tips of Pisum. Am J Bot 53:970-976 (1966). 17. Van't Hof J: Studies on the relationships between cell population and growth kinetics of root meristems. Exp Cell Res 46:335-347 (1967). 18. Van't Hof J: Experimental procedures for measuring cell population kinetic parameters in plant root meristems. In: Prescott DM (ed). Methods in Cell Physiology, vol. 3, pp. 95-117. Academic Press, New York (1968). 19. Van't Hof J, Lamm, SS: Single-stranded replication intermediates of ribosomal D N A replieons of pea. EMBO J 10:1949-1953 (1991). 20. Van't Hof J, Hernfindez P, Bjerknes CA, Kraszewska EK, Lamm SS: Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient Eeo RI sites in replicating rRNA genes. Plant Mol Biol 8:133-143 (1987). 21. Wagenaar EB: High mitotic synchronization induced by 5-aminouracil in root cells ofAllium eepa L. Exp Cell Res 43:184-190 (1966).
377
Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis Jack Van 't Hof and Susan S. Lamm
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA Received 18 November 1991; accepted in revised form 14 May 1992
Key words: replication, origin, rDNA, 5-aminouracil, Pisum sativum, synchrony Abstract
The time course of replication of the 9 kb ribosomal D N A repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.
Introduction
Previously we located the replication origin of the 9 kb ribosomal D N A repeats (rDNA) of pea in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene [9]. The method used to localize the origin, however, did not provide the time-dependent evidence expected of replication initiation followed by fork movement within the replicon. Here we show that indeed replication starts in the r D N A fragment in which we found the origin to be located, and then proceeds to the adjoining r D N A sequences within the repeat. This process is detectable using less than 0.5 #g ofgenomic D N A from root meristems in which 6 ~o or less of the cells are replicating their DNA. Materials and methods
[ 18, 19]. Meristematic cells of primary roots were synchronized by suspending roots of seedlings for 12 h at 20 °C in an aerated solution containing nutrient salts and 5-aminouracil (0.2mg/ml; Sigma A-4005). 5-aminouracil temporarily blocks dividing cells in late S phase. The blockage is removed when seedlings are transferred to fresh nutrient salt solution containing no inhibitor. After treatment meristematic cells recover and divide synchronously within 7 h. To determine when and how many cells divided, 2 to 3 mm tips of primary roots were fixed with 3 parts methanol, 1 part glacial acetic acid, hydrolyzed with 5N HC1 at 22 °C for 30 to 45 min, stained either by the Feulgen method or with toluidine blue, and squashed on microscope slides. Dividing cells were counted from random samples on the slides, 1000 cells per meristem, three meristems per sample.
Ceil synchronization
Single-cell autoradiography
Pea seeds (Pisum sativum cv. Alaska) were germinated and grown as previously described
Cells in S phase at the 6th h of recovery were labeled for 1 h with 3H-thymidine (3 #Ci/ml; sp.
378 act. 55 Ci/mmol) by immersing the primary roots of seedlings in 250 ml of nutrient solution containing the isotope. After transfer to nonradioactive medium, root tips were fixed at hourly intervals up to 13 h, stained by the Feulgen method, squashed on microscope slides and processed for autoradiography by published procedures [16, 17]. The autoradiograms were scored for percent labeled division figures to determine when and how many of the radioactive cells divided. All other methods used in these experiments are as described by Van't Hof and Lamm [19] unless noted otherwise in the figure legends.
Results and discussion
Design of the experiment At least three factors must be considered in designing an experiment with a high probability of detecting replication initiation in pea root cells. One factor is cytological. Since r D N A is replicated throughout S phase [20], r D N A from asynchronously dividing cells will yield replicons in all stages of replication: initiation, chain elongation and termination. To enrich for replicons in the initiation stage, we used an inhibitor, 5-aminouracil, to block cells in late S phase. Upon removal of the blockage, we anticipated that cells still in S phase would initiate replication of their genome and complete the process while other cells, such as those of the quiescent center, would begin D N A replication in response to the effects of 5aminouracil [4, 5]. Given a situation where cells can be manipulated so they begin replication synchronously, a second factor must be considered. This factor is a means of distinguishing between replicative intermediates and non-replicating molecules. In the present experiments we used twodimensional gel electrophoresis because this method distinguishes between replicative and non-replicative molecules and also separates them [ 1, 2, 3, 12, 19]. The final factor to consider is the probes used to identify which sequence within the r D N A repeat functions as a replication origin. The probes chosen should hybridize with
specific sequences located at different positions within the repeat.
Division of synchronized cells Root meristem cells when treated with 5aminouracil continue D N A synthesis at a reduced rate [16] and are blocked in late S phase of the cell cycle [8, 13, 14, 15, 16]. The blockage is temporary, however, as cells divide eventually even in the presence of the inhibitor [13, 21, J. Van't Hof, unpublished results]. When in place, the block prevents cells from proceeding from S to G2 phase. Thus, during the first 4 h of treatment, cells continue to divide in decreasing numbers and after 12 h of treatment meristems have few, if any, dividing cells. Recovery commences when the inhibitor is removed. At the 7th hour of recovery cells divide reaching 35~o of the total number of cells in the root tip by the 8th hour (Fig. 1). Cell division remains near this level for 1.5 h (9.5 h in Fig. 1) and then decreases to control level at 12 h.
50 45 _~
40
o
35
"0
25 "O
.
20
c
o
15
a.
10~
untreated control
3 ,I'
0
~' Hours
~ after
H " ",,,,,,,
1'o
¢1
1'~
1~
5-AU t r e a t m e n t
Fig. 1. The percent dividing cells in the meristematic tip of primary roots of pea seedlings expressed as a function of time after a 12 h treatment with 0.2 mg/ml 5-aminouracil. Open triangles, non-radioactive dividing cells; dotted line, radioactive dividing cells labeled with 3H-thymidine for 1 h 6 hours after 5-aminouracil treatment; dashed line (control), dividing cells in root tips neither treated with 5-aminouracil nor labeled with isotope.
379 The 1.5 h span in which the number of dividing cells remains relatively constant is roughly equivalent to the duration of mitosis in pea [17]. Thus, cells in prophase at 8 h will be in anaphase and telophase at 9.5 h. As this cohort completes division the number of dividing cells decreases. Following this first cohort, a smaller one begins to divide at 9.5 h and it too, completes division in about 1.5 h. Among the smaller cohort are cells that incorporate ~H-thymidine at the 6th hour of recovery (Fig. 1). These cells represent 6~o or less of the total number of dividing cells in the meristem. We do not know the source of the cells in our experiment but the work of Clowes indicates that they very likely originate from the quiescent center [4, 51.
work with electron microscopy show 83, 92 and 94~o of the molecules in spots A, B and C, respectively, to be linear. The probes, 790, 1.3 and 1.6, are homologous to r D N A sequences located at increasing distances from the origin. Probe 790 hybridizes to the replication origin and two contiguous subrepeats upstream from the origin, probe 1.3 to 1.3 kb located ca. 1 kb to the right of the origin and contains ARS consensus sequences [9] and probe 1.6 to coding sequences located about 3 kb downstream to the right of the origin. Since the r D N A repeats are tandem, probe 1.6 also hybridizes to sequences about 4 kb upstream to the left of the origin.
Spots A, B and C are transitory Detection of single-stranded replication fragments of rDNA Ribosomal D N A of pea, either digested with Eco RI or double-digested with Eco RI and Hind lII and separated by neutral-neutral twodimensional electrophoresis, produces three spots of single-stranded replicative intermediates [ 19]. In the second dimension these spots migrate as an arc above another, dominant, arc of duplex molecules. The r D N A sequences in the spots, the length of the single-stranded chains they contain and the probes to which they hybridize are given in Table 1. More recent data from preliminary Tablel. Summary of molecular characteristics of spots A, B, and C containing single-stranded replication intermediates of r D N A of pea
Spot
Size (kb)*
Hybridization to probes
rDNA sequences
790
1.3
1.6
A
9
+
+
+
B
5
+
-
+
C
3.6
+
+
-
spacer, 18S, 5.8S, 25S spacer, 25S spacer, 18S
Data from Van't Hof and Lamm [19]. * Determined by alkaline gel electrophoresis.
When replication is reduced or stopped the spots are either absent or only slightly visible when hybridized to specific probes. When replication is resumed, the spot containing the replication origin should appear first. Later on, after replication is beyond the initiation stage, all three spots should be detectable. Evidence that these changes occur is shown in Fig. 2. The autoradiograms are of fragments of r D N A produced by double digestion with Eco RI and Hind III. Each blot was probed with 790, a sequence that hybridizes to all three spots [19]. The two arcs in the autoradiograms reflect the different migration rates of replicative and nonreplicative r D N A fragments in the seconddimension gel. The darker lower arc contains duplex r D N A fragments that are not replicating [19]. The upper arc has single-stranded replication intermediates seen as three spots, labeled A, B and C. Note that each autoradiogram has spots but their size and hybridization signal differ with the source of rDNA. The signals are weak if the r D N A is from cells given 3 h to recover (Fig. 2a) strong, if the r D N A is either from cells given 12 h of recovery (Fig. 2b) or from untreated (control) asynchronously dividing cells (Fig. 2c). We interpret these results to indicate that little r D N A replication occurs 3 h after 5-aminouracil treatment
380
Fig. 2. Identification of replication intermediates of ribosomal D N A of pea root cells. Autoradiograms of Southern blots of ribosomal D N A double-digested with Eco RI and Hind III. Fragments were separated by two-dimensional gel electrophoresis run under neutral conditions in both directions and probed with 790. a) r D N A fragments from meristematic cells 3 h after being treated for 12 h with 0.2 mg/ml 5-aminouracil; b) fragments from cells 12 h after treatment; c) fragments from untreated (control) meristematic cells. A, B and C are spots of replicative intermediates on an arc located above a darker arc formed by linear duplex r D N A fragments. The numbers, in kb, refer to duplex rDNA fragments. Direction of 1st dimension (0.5% agarose; 0.66 V/cm, 19.5 h), right to left; 2nd dimension, (1.75% agarose, a; 1.5% agarose, b and c; 10 V/cm, 2.5 h), top to bottom.
Fig. 3. Autoradiograms of a Southern blot of ribosomal D N A Eco RI-Hind III fragments from pea root meristems separated by two-dimensional gel electrophoresis under neutral conditions in both directions. The blots were probed, stripped and probed again sequentially with 790, 1.3 and 1.6. The probes are noted above the autoradiograms, a), b) and c) fragments from cells 6 h after treatment with 5-aminouracil; d), e) and f) fragments from cells 8 h after treatment. Spots A, B and C, numbers and direction of migration are as described in Fig. 2; 1st dimension 0.5% agarose, 0.66 V/cm, 19.5 h; 2rid dimension 1.75% agarose, 10 V/cm, 2.5 h.
and that it is resumed by the 12th hour. Replication began, therefore, some time between the 3rd and 12th h after treatment.
Evidence that r D N A replication begins 6 h after treatment is shown in Fig. 3, the top row of panels. The autoradiograms in these panels are of
381 the same blot of r D N A probed, stripped and probed again sequentially with the three probes noted above the panels. Note that probe 790 hybridized strongly only to spot C (Fig. 3a). Probe 1.3, likewise, hybridized only to spot C but the signal is weak (Fig. 3b). On the other hand, probe 1.6 failed to hybridize to any spot (Fig. 3c). It is significant that probe 790 gave a strong signal with spot C and weak signals with spots A and B. This probe hybridizes to all spots, if they are present on the blot (Figs. 2b and 2c). The weak hybridization to spots A and B, therefore, indicates that sequences contained in these spots are not replicating at 6 h. If replication begins in sequences contained in spot C, then as time passes active replication forks are expected to move to sequences contained in spots A and B. As seen in Fig. 3, the bottom row of panels, an additional 2 h of recovery does produce the expected result. Autoradiograms of blots of ribosomal D N A from cells 8 h after treatment show three spots when probed with 790 (Fig. 3d), two (A and C) when probed with 1.3 (Fig. 3e) and two (A and B) when probed with 1.6 (Fig. 3f). Taken together, the autoradiograms in Fig. 3 provide evidence that the replication of the r D N A repeats follows a temporal order and that replication begins in sequences contained in spot C. The sequences in spot C are those of the spacer region including the subrepeats. Among the subrepeats, approximately 1.5 kb downstream from the 3' end of the 25S gene, is a 209 base sequence identified previously as the replication origin [9]. Finally, it should not be overlooked that the results presented here were obtained using less than 0.5 #g of genomic D N A per gel. Of this DNA, approximately 0.08~o or 0.4 ng is rDNA. This is enough r D N A to detect replication intermediates, given that there are about 3900 copies of ribosomal genes in pea var. Alaska [7, 10]. Previously, we demonstrated that the replication intermediates are single-stranded and products of serial replication displacement loops [ 19]. Since these intermediates are detectable with as little as 0.5/~g of genomic DNA, replication by serial displacement loops is likely the most common, but
not necessarily the only, mechanism of r D N A replication in pea. The meristem is a complex tissue composed of several distinctive cell populations [6] a feature that distinguishes it from single cell systems such as yeast and cultured mammalian cells. The response of cells in the meristem to 5-aminouracil treatment, therefore, may differ from that expected of single cell systems. For example, in the meristem, mitotic inhibition of most cells is the signal for others in the quiescent center to initiate D N A replication and divide [4, 5]. Further, meristematic cells may differ from yeast cells in the means used to replicate rDNA. Yeast cells replicate r D N A unidirectionally via leading and lagging strands [3, 12] while in pea the data support replication by serial displacement loops [ 19]. On the other hand, in view of the variety of mechanisms used by viruses to replicate their genome [ 11 ] it is possible that higher plants use more than one mechanism to replicate their genome.
Acknowledgements Research was supported by the Office of Health and Environmental Research, US Department of Energy.
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