Plant Molecular Biology 5: 353-361, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht - Printed in The Netherlands
E x t r a c h r o m o s o m a l D N A o f pea-root r i b o s o m a l genes
(Pisum sativum)
has repeated s e q u e n c e s and
E. K. Kraszewska, C. A. Bjerknes, S. S. Lamm & J. Van 't H o f Biology Department, Brookhaven National Laboratory, Upton, N Y 11973, US.A.
Keywords: cell differentiation, restriction enzyme digestion, Southern blotting analysis
Summary Restriction endonuclease digestion and Southern blotting procedure were used to determine differences between extrachromosomal, nuclear, plastid, and mitochondrial DNAs from meristematic cells of cultured pea roots. Extrachromosomal and nuclear DNA are highly methylated and neither DNA is homologous to plastid or mitochondrial DNA. Hybridization of extrachromosomal DNA to nuclear DNA indicated that extrachromosomal DNA differed quantitatively from total nuclear DNA in repetitive sequences. Cloned rDNA showed that extrachromosomal DNA contains rRNA genes but the hybridization signal indicated that the copy number was less than that expected if the molecules were amplified. These and cytological findings suggest that extrachromosomal DNA is involved in or a product of genomic changes associated with the onset of differentiation by precursor cells of vascular parenchyma and the root cap.
Introduction When meristematic root-tip cells stop dividing they arrest in the G~ or G2 phases of the cell cycle and differentiate with a corresponding 2C or 4C amount of nuclear DNA (7, 8, 25). The mechanism responsible for the transition of a cell from a proliferative to a differentiated state is unknown but recent work on chromosomal DNA replication in cultured pea roots suggests that the excision of certain nascent replicons ( - 5 4 kb) from the chromosomal duplex constitutes an initial step toward differentiation of certain root-tip cells (26, 27, 28). Three observations support this suggestion. First, velocity sedimentation in alkaline sucrose gradients show that not all nascent replicons mature to chromosomal-size DNA, i.e., reach a singlestranded size of 6 to 8x108 daltons (26). Some replicons persist as free molecules up to three days after being replicated and remain associated with the cells in which they are formed as these cells are
displaced from the meristem to the elongation zone (26). Second, the free nascent DNA molecules are produced by these cells after they are temporarily arrested in late S phase prior to differentiating from G 2 phase (27). Third, the cells displaying these phenomena are the precursors of vascular parenchyma and root-cap cells (28). A common thread running through these observations is that specific cells are temporarily impeded during late S phase, that these cells differentiate from G 2 phase, and that these cells have persistent replicon-sized molecules that do not mature to chromosomal size. The free DNA molecules, called extrachromosomal DNA (exDNA), are the topic of this paper. Here we present evidence that exDNA contains rRNA genes and other repetitive sequences that differ quantitatively relative to those found in nuclear DNA.
354 Materials and methods
Culture of root tips Seed of Pisum sativum (var. Alaska) were surface sterilized with Clorox, washed with distilled water, and aseptically germinated in Petri dishes (150 x 20 mm) that contained three layers of Whatman no. 1 filter paper moistened with distilled H20. After 4 days, seedlings with primary roots 2 . 5 - 4 cm long were selected, the terminal 1-1.5 cm detached, suspended in 50 ml of White's medium and cultured in flasks at 22_+1 °C on a reciprocal shaker at about 1 cycle/sec. Seed germination and root culture were carried on in the dark and only exposed to light when the cultures were examined. Nuclei were isolated from two sources, from the meristematic tip of roots cultured continuously in medium with sucrose and from tips of roots cultured without sucrose. The former had dividing cell randomly distributed in the cell cycle while the latter has little to no mitosis and the meristematic cells are accumulated in the G1 and G2 phases of the cell cycle (24, 25). These two sources represent different physiological conditions and offer a control for the character of the extracted exDNA molecules.
Isolation of exDNA fragments Batches of 25 roots, from a total of 900 roots, were washed with ice-cold sodium phosphate buffer (0.132 M, pH 6.8), treated for 30 sec with 2°7o formaldehyde in phosphate buffer, washed three times with liberal amounts of ice-cold buffer and the 3 mm meristematic tip cut from the root. Each tip, placed on a subbed microscope slide (slides coated with 0.507o gelatin - 0.0507o Crk(SO4)2 • 12H20 and air dried) in about 25/A of buffer, was covered with another slide and tightly pinched between the thumb and index finger, forcing a drop of nuclei at the slide's edge. Drops of nuclear suspension from 25 root tips collected in a chilled 1.5 ml Eppendorf tube were centrifuged at 3 0 0 x g for 5 min at 4 °C to pellet the nuclei. After removal of the supernatant, 100/~1 of selfdigested pronase (10 mg/ml) were added to each pellet and the mixture incubated at room temperature for 1 h. The nuclei were lysed and the
DNA fragments extracted as described by Hirt (11). This involved the careful addition of 150/~1 of 1.2°70 SDS, 0.02 M EDTA, pH 7.5, gently bringing the tube to a horizontal position once and allowing lysis to occur at room temperature for 20 min. The solution adjusted to 1.3 M NaC1 by addition of 75/zl of 5 M NaC1 was mixed by gently rocking the tube at a horizontal position 10 times and placed in ice in a cold room (4 °C) for at least 8 h. The precipitated material was pelleted by centrifugation in the cold at about 13000xg for 15 min in a Beckman microfuge, the tubes removed and flicked with the index finger to dislodge any precipitate attached to the side of the tubes, centrifuged again for 15 min, and placed in ice for 30 min to harden the pellet. The supernatants were combined in a polyallomer tube and the DNA pelleted by centrifugation in a Beckman SW 50.1 rotor at 50000 rpm at 2 °C for 150 min. The DNA was resuspended by vortexing and the solution transferred to a 1.5 ml Eppendorf tube. The polyallomer tube was washed with an additional 100/xl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), vortexed and the wash added to the DNA solution. The DNA was incubated with pancreatic RNase (10/zl of 1 mg/ml RNase per 100/zl of solution) for 1 hr at 37 °C, the solution was extracted with an equal volume of TE-saturated phenol, and the DNA precipitated by the addition of 2 volumes of ice-cold 95070 ethanol and storing at - 2 0 ° C overnight. The precipitate was pelleted by centrifugation in a Beckman microfuge at 13000xg for 30 min, the supernatant removed, the pellet washed with 67070 ethanol chilled to - 2 0 ° C , allowed to stand in ince for 30 min, and again spun at 13000xg for 15 rain. The pellet was dried by lyophilization, dissolved in 1/4 strength TE buffer and dialyzed on a Millipore VSWP filter for 1 h at 22°C against 1/4 strength TE and lyophilized.
Separation of methylated exDNA fragments ExDNA was digested with the restriction endonuclease Hpa II and the fragments separated by gel electrophoresis in a low melting temperature agarose. Fragments larger than 4.3 kb were recovered from the stained gel by melting and phenol extraction (19).
355
Extraction of total D N A The method of Burr and Burr (4) was used to extract total D N A from cultured root tips grown in medium with sucrose for 3 days.
stained gels were exposed to 2 min of 254 nm UV light. After transfer the gels were stained and checked to assure the transfer was complete. The radioactivity applied to each blot ranged from 2 . 2 - 3 . 0 x 1 0 6 cpm and exposure to x-ray film varied from 6 to 48 h depending on the experiment.
Isolation of mitochondrial and plastid DNA The method of Kolodner and Tewari (15, 16, 17) was used to obtain organelle D N A from roots cultured with sucrose for 3 days. The procedure was scaled downward to accommodate small amounts of tissue.
Pea-cDNA clone The plasmid, pHA-1 a derivative of pACYC184, containing a 9 kb Hind III insert with the 18S, 5.8S, and 25S r R N A genes was a gift from Dr R. E. Cuellar (5). The plasmid was cloned in E. coli HB101 and the insert isolated by published procedures (19).
Results
Isolation of nuclei from cultured root tips When dealing with D N A molecules smaller than chromosomal-size it is necessary to minimize random breakage during the isolation procedure. The best approach is to extract D N A from clean, unbroken nuclei. Our method of isolating nuclei from root tips meets this requirement (Fig. 1). The nuclei seen in Fig. la are from a single 3 m m root tip of
[32P]-labeled probes Mitochondrial DNA, plastid DNA, exDNA, total D N A and r D N A were labeled by nick translation to a specific activity of 2 - 4 x 108 cpm/~g using a kit purchased from Bethesda Research Laboratories.
Restriction enzyme digestion Restriction enzymes obtained from Bethesda Research Laboratories were used according to the supplier's specifications.
Gel electrophores& D N A fragments were separated by electrophoresis in 0.6o70 agarose gels using TAE buffer (0.04 M T r i s . HC1, 0.02 M glacial acetic acid, 1 mM EDTA, p H 8.0).
Southern blot hybridization Fig. 1. Nuclei isolated from the 3-mm tip of pea roots, la, phoD N A transfer to Zeta-Probe (Bio-Rad Laboratories), prehybridization, hybridization, washing and strip-wash conditions followed the method o f Gatti et al. (9). Prior to transfer ethidium bromide
tomicrograph of Feulgen-toluidine blue stained nuclei isolated from a single root tip. Ib, an electronmicrographof pelleted isolated nuclei prepared by Dr M. C. Ledbetter of Brookhaven National Laboratory. The average diameter of round nuclei is 8/~m.
356 pea, and t h o u g h p h o t o g r a p h e d at low magnification, they appear unbroken and free o f cytoplasmic tabs. This is true also for nuclei viewed with the electron microscope as seen in Fig. lb. Such nuclei are a proven source o f unsheared, undegraded chrom o s o m a l D N A . They yield nascent molecules o f 6 to 8 x l 0 s daltons under alkaline conditions (26) and double-stranded molecules that often exceed 1 m m or 2x109 daltons (29). The large size o f these molecules is attributable to gentle isolation o f nuclei and to the inactivation o f nucleases by a short pretreatment with 2°7o buffered formaldehyde (J. B. Schvartzman, personal communication).
Separation of exDNA fragments from organelle DNA O u r m e t h o d o f extracting e x D N A does not exclude some organelle DNAs. To separate e x D N A from organelle D N A s we exploited the fact that organelle D N A has fewer methylated bases than nuclear D N A (2, 3, 10, 22, 23). This difference is detectable in D N A digested by the methyl-sensitive
restriction endonuclease, H p a II. Separation o f the H p a II fragments by gel electrophoresis shows that the enzyme cleaves plastid and mitochondrial D N A s to fragments smaller than 4.3 kb (Fig. 2, lanes 1 and 5, respectively). E x D N A and total D N A , on the other hand, are cleaved to fragments larger than 4.3 kb (Fig. 2, lanes 2 and 3, respectively). Hybridization experiments show that the smaller fragments, present in digested e x D N A but absent in total D N A , are h o m o l o g o u s to organelle D N A . Labeled plastid D N A hybridized to these smaller fragments (Fig. 2a, lane 2) giving a banding pattern identical to that o f digested plastid D N A (Fig. 2b, lane 1). Labeled mitochondrial D N A likewise hybridized to H p a II fragments in the digested e x D N A and these are smaller than 4.3 kb (Fig. 2c, lane 4). There are other points worth noting about this experiment. One concerns the low a m o u n t o f hybridization o f plastid D N A to e x D N A fragments o f approximately 23 kb (Fig. 2a, lane 2). The source o f this light signal is obscure. It may indicate a low level o f h o m o l o g y between some e x D N A
Fig. 2. Ethidium-bromide stained agarose gels of plastid, mitochondrial, exDNA, and nuclear DNA digested with Hpa I1 and autoradiograms of blotted DNAs hybridized to either plastid or mitochondrial DNA probes. Stained gel on the left: lane 1, plastid DNA; lane 2, exDNA; lane 3, nuclear DNA. Approximately 0.1 /~g of DNA was loaded per well. Autoradiogram (a), hybridization of [32p]-plastid DNA to blotted DNAs of the gel on the left; the film was exposed for 30 h. Autoradiogram (b), same as (a) except the film was exposed for 9 h. Stained gel on the right: lane 4, exDNA~ lane 5, mitochondrial DNA. Each lane contained approximately 0.12 ~g of DNA. Autoradiogram (c) hybridization of [32p]-mitocbondrialDNA to blotted DNAs of gel pictured on the right. Film was exposed for 6 h. kb, kilobases, noting sizes of marker DNA.
357 fragments and plastid D N A or it may reflect a small amount of nuclear D N A present in the plastid D N A preparation. Whatever its source, the hybridization is so weak compared to that of fragments less than 4.3 kb that its presence is considered insignificant to the present work. Another point is significant. It concerns the absence of hybridization of either the plastid or mitochondrial D N A probes to total D N A (Fig. 2a, lane 3; data using the mitochondrial probe is not shown). The absence of hybridization under our experimental conditions demonstrates that total D N A is equivalent to nuclear D N A and we shall consider it as such hereafter. Finally, the stained gels show that more than 90°70 of the D N A in the exDNA extract has no homology to either plastid or mitochondrial D N A (Fig. 2, lanes 2 and 4).
Homology o f exDNA to nuclear D N A The lack of hybridization of organelle DNAs to most of the H p a II fragments in exDNA indicates that exDNA is of nuclear origin, but we did not know if exDNA represented a few or many nuclear sequences. Therefore, we labeled exDNA H p a II fragments, freed of organelle DNAs, and probed
six separate digests of nuclear D N A to detect homologous sequences. The range of homology is extensive as seen on the autoradiogram shown in Fig. 3a. The extent of hybridization precluded the detection of any specificity of the exDNA. To see differences between exDNA and nuclear sequences it was necessary to hybridize labeled nuclear D N A to the same blot and compare the pattern of hybridization with that produced with exDNA. The autoradiogram using the nuclear D N A probe is shown in Fig. 3b and it has a different pattern than that produced using the exDNA probe (Fig. 3a). For example, exDNA hybridized less to a 7 kb Bgl 1 fragment (Fig. 3a, lane 4) than did nuclear D N A (Fig. 3b, lane 4). A more exacting demonstration of differences, however, was obtained from densitometric tracings of the autoradiograms (Fig. 4). The tracings, seen as sets of two one superimposed upon the other, represent individual measurement of the first three lanes of the autoradiograms. Figure 4a has tracings of hybridization to nuclear D N A digested with Eco R1 (lane 1 of the autoradiograms). The rough dotted line is that of the exD N A probe, the smooth line, is that of the nuclear D N A probe. The tracings show that exDNA hybridized less than nuclear D N A to 7 and 4.3 kb fragments, and more to fragments of 4.3, 3.3, 1.9 and
Fig. 3. Ethidium-bromide stained agarose gel of nuclear DNA digested with Eco RI, lane 1; Barn HI, lane 2; Hind IlI, lane 3; Bgl I, lane 4; Xho I, lane 5; Hinc II, lane 6. Each lane contained approximately 0.4 p.g of DNA. Autoradiogram (a): hybridization of [32p]_ exDNA, 2.5 × 106 cpm, to blotted DNA. Autoradiogram (b): hybridization of [32p]-nuclear DNA, 2.5 × 106 cpm, to blotted DNA. To achieve approximately the same grain density (a) was exposed 24 h, (b) 48 h. kb, kilobases, noting sizes of marker DNA.
358 e
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I'~ el
I
e
kb e~ •
e.e.
.~
o I
•
0
Q e. ~ •
o .
(¢)
(a) (b)
Y
Fig. 4. Densitometric tracings of each of the first three lanes of the autoradiograms shown in Fig. 2. The rough dotted lines are tracings of grain densities of exDNA hybridized to nuclear DNA. The smooth lines are tracings of nuclear DNA hybridized to nuclear DNA. (a), hybridization to nuclear DNA digested with Eco RI, (b), hybridization to nuclear DNA digested with Bam HI, (c), hybridization to nuclear DNA digested with Hind III. kb, kilobases, noting sizes of marker DNA.
1.5 kb. The second set of tracings measures hybridization to DNA digested with Bam HI (Fig. 4b). It shows that exDNA hybridized less than nuclear DNA to fragments of 10.6 and 4.6 kb, more to those of 5.4 and 3.6 kb and more to six fragments smaller than 3.6 kb. Lastly, Fig. 4c shows tracings of hybridization to DNA digested with Hind III (lane 3 of each autoradiogram). In this case, the most notable difference is the amount of hybridization to a 9.6 kb fragment. Relative to the peak density at 5.8 kb, seen in each autoradiogram, the amount of exDNA that hybridized to the 9.6 kb fragment is 43°70 less than that of the nuclear DNA. E x D N A contains ribosomal genes
Extrachromosomal rDNA occurs frequently in unicellular eukaryotes (18), slime mold (31, 33), and amplified rDNA in oocytes is always extrachromosomal (18). A low level of rDNA amplification is reported also to occur in roots of Allium cepa (1). These observations motivated us to determine if exDNA of pea roots likewise had rRNA se-
quences. For the experiment we used the plasmid pHA-1, a derivative of pACYCI84, as a source of rDNA (5, 14). The 18S, 5.8S, and 25S rRNA genes of pea are located on a single 9 kb Hind III inserted into the plasmid. The isolated, labeled insert was used to probe Hind III digests of nuclear DNA and exDNA from dividing and non-dividing root-tip cells. Figure 5 shows that the probe is homologous to a single 9 kb fragment in the digests of each DNA indicating not only the presence of rDNA sequences on exDNA but also that the sequences are confined to the same fragment as nuclear DNA. The importance of this finding should not be overlooked. It demonstrates that exDNA extracted from cells subjected to different physiological conditions is identical, and that exDNA is not a product of random DNA breakage. We repeatedly detect rDNA sequences on exDNA and they are always on a 9 kb Hind III fragment. This indicates that the rRNA genes are on a contiguous strand of exDNA that is never broken separating the genes. Also, the frequency of rRNA genes in the pea genome is 8× 10 -3 making it improbable on a random basis
359
Fig. 6. Autoradiogram of dot blot of exDNA from dividing
meristems, row (a); exDNA from non-dividing meristems, row (b); nuclear DNA, row (c). The dots werehybridized with cloned [32p]-rDNA. Columns (1), (2), and (3), respectively, 0.1, 0.01, and 0.001 #g of DNA blotted. The film was exposed for 6 h. Fig. 5. Autoradiogram of cloned [32P]-rDNA hybridized to
exDNA from dividing meristems, lane 1; exDNA from nondividing meristems, lane 2; nuclear DNA, lane 3. Each lane contained about 0.1 /zg of DNA digested by Hind II1. The film was exposed for 6 h. Ori, gel origin; kb, kilobases.
that the genes would consistently be extracted as exDNA. Given a genome size of 4.6×10-12 g (20) and 3.9x103 r R N A genes per genome (6, 13), there are about 8 × 107 r R N A genes per 0.1 /~g of pea DNA. E x D N A molecules are approximately 54 kb and 0.1 #g of exDNA represents 1.8 × 109 molecules. I f each molecule has r R N A genes, exDNA would be 400 times richer in these sequences than nuclear DNA. Dot blots of exDNA and nuclear D N A hybridized with labeled cloned rDNA, however, show that exDNA from either dividing or nondividing cells has fewer ribosomal sequences than nuclear D N A (Fig. 6). Scintillation counts of the dot blots with 0.1/zg exDNA were 15 to 25% less than that of nuclear DNA. Assuming that 0.1 /~g of nuclear D N A has 8>(107 r R N A genes, the lower radioactivity hybridized to exDNA corresponding to approximately 6.4 >( 107 genes, 1.7 x 109 less than that expected if each molecule had r R N A sequences. It is unlikely, therefore, that the ribosomal sequences in exDNA are amplified.
Discussion In this paper evidence is presented that exDNA molecules of cultured pea-root cells represent repetitive sequences and r R N A genes in relative
amounts that differ from those found in nuclear DNA. Repetitive sequences, in this instance, is used according to H u m m e l et al. (12) who defined them as sequences given rise to visible bands on a gel after electrophoresis of D N A digested by restriction endonucleases. Unique sequences escaped detection in our experiments because the amounts of D N A used in the Southern blots was 0.4 #g or less. The results of these experiments support our earlier conclusion that exDNA represents certain replicons of late replicating D N A that are excised from the chromosomal duplex of specific cells while they are delayed in late S phase prior to differentiating from G 2 phase (27). Recent work shows that these ceils undergo a pronounced change in nuclear shape during the transition from a meristematic to a differentiated state (28). When finishing replicating their DNA, the cells have spherical or round nuclei, and produce exDNA. U p o n completion of D N A replication, their nuclei become elongated or oblong often reaching lengths five times the diameter of the original sphere. This transformation clearly involves structural changes in the chromosomal duplex and we believe these changes include removal of segments (exDNA) from the chromosomal DNA. Whether or not the transformation also involves amplification of certain sequences, of which r D N A is excluded, remains to be determined. Our attempts, though not exhaustive, to detect homology to transcripts other than r R N A have failed, but the possibility still exists that exDNA has other genes. It is also possible that the production of exDNA in pea-root cells is a phenomenon analogous to
360 that observed in developing cells of Tetrahymena (32) and nematodes (21) where differentiation is associated with chromosomal breakage and gene rearrangements. The production of exDNA likewise is consistent with Varshavsky's idea that extrachromosomal copies of nuclear DNA are produced by 'misfiring' of certain replicons (30). 'Misfiring' refers to the additional replication of replicons above that which occurs normally during each round of DNA replication. If multiple 'misfirings' only involve a single, specific replicon, amplification results, but an additional one or two 'misfirings' in several different replicons would produce extrachromosomal DNA of the variety seen in pea. Judging from the work reviewed by Varshavsky and from that on exDNA in pea, the production of replicon-sized nuclear DNA free of the chromosomal duplex is a mechanism involved in the transition of a cell from a proliferative to a differentiated state.
Acknowledgement Research supported by the U.S. Department of Energy and the Monsanto Agricultural Chemical CO.
9. 10.
11. 12.
13. 14.
15.
16.
17.
18. 19.
20.
21.
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361 29. Van 't Hof J, Kuniyuki A, Bjerknes CA: The size and number of replicon families of chromosomal DNA of Arabidopsis thaliana. Chromosoma 68:269-285, 1978. 30. Varshavsky, A: On the possibility of metabolic control of replicon 'misfiring': Relationship to emergence of malignant phenotypes in mammalian cell lineages. Proc Natl Acad Sci USA 78:3673- 3677, 1981. 31. Vogt VM, Braun R: Structure of ribosomal DNA in Physarum polycephalum. J Mol Biol 106:567-587, 1976.
32. Yao M-C: Ribosomal RNA gene amplification in Tetrahymena may be associated with chromosomal breakage and DNA elimination. Cell 24:765-774, 1981. 33. Zellweger A, Ryser U, Braun R: Ribosomal genes of Physarum: Their isolation and replication in the mitotic cycle. J Mol Biol 64:681-691, 1972. Received 30 May 1985; in revised form 21 August 1985; accepted 27 August 1985.
E x t r a c h r o m o s o m a l D N A o f pea-root r i b o s o m a l genes
(Pisum sativum)
has repeated s e q u e n c e s and
E. K. Kraszewska, C. A. Bjerknes, S. S. Lamm & J. Van 't H o f Biology Department, Brookhaven National Laboratory, Upton, N Y 11973, US.A.
Keywords: cell differentiation, restriction enzyme digestion, Southern blotting analysis
Summary Restriction endonuclease digestion and Southern blotting procedure were used to determine differences between extrachromosomal, nuclear, plastid, and mitochondrial DNAs from meristematic cells of cultured pea roots. Extrachromosomal and nuclear DNA are highly methylated and neither DNA is homologous to plastid or mitochondrial DNA. Hybridization of extrachromosomal DNA to nuclear DNA indicated that extrachromosomal DNA differed quantitatively from total nuclear DNA in repetitive sequences. Cloned rDNA showed that extrachromosomal DNA contains rRNA genes but the hybridization signal indicated that the copy number was less than that expected if the molecules were amplified. These and cytological findings suggest that extrachromosomal DNA is involved in or a product of genomic changes associated with the onset of differentiation by precursor cells of vascular parenchyma and the root cap.
Introduction When meristematic root-tip cells stop dividing they arrest in the G~ or G2 phases of the cell cycle and differentiate with a corresponding 2C or 4C amount of nuclear DNA (7, 8, 25). The mechanism responsible for the transition of a cell from a proliferative to a differentiated state is unknown but recent work on chromosomal DNA replication in cultured pea roots suggests that the excision of certain nascent replicons ( - 5 4 kb) from the chromosomal duplex constitutes an initial step toward differentiation of certain root-tip cells (26, 27, 28). Three observations support this suggestion. First, velocity sedimentation in alkaline sucrose gradients show that not all nascent replicons mature to chromosomal-size DNA, i.e., reach a singlestranded size of 6 to 8x108 daltons (26). Some replicons persist as free molecules up to three days after being replicated and remain associated with the cells in which they are formed as these cells are
displaced from the meristem to the elongation zone (26). Second, the free nascent DNA molecules are produced by these cells after they are temporarily arrested in late S phase prior to differentiating from G 2 phase (27). Third, the cells displaying these phenomena are the precursors of vascular parenchyma and root-cap cells (28). A common thread running through these observations is that specific cells are temporarily impeded during late S phase, that these cells differentiate from G 2 phase, and that these cells have persistent replicon-sized molecules that do not mature to chromosomal size. The free DNA molecules, called extrachromosomal DNA (exDNA), are the topic of this paper. Here we present evidence that exDNA contains rRNA genes and other repetitive sequences that differ quantitatively relative to those found in nuclear DNA.
354 Materials and methods
Culture of root tips Seed of Pisum sativum (var. Alaska) were surface sterilized with Clorox, washed with distilled water, and aseptically germinated in Petri dishes (150 x 20 mm) that contained three layers of Whatman no. 1 filter paper moistened with distilled H20. After 4 days, seedlings with primary roots 2 . 5 - 4 cm long were selected, the terminal 1-1.5 cm detached, suspended in 50 ml of White's medium and cultured in flasks at 22_+1 °C on a reciprocal shaker at about 1 cycle/sec. Seed germination and root culture were carried on in the dark and only exposed to light when the cultures were examined. Nuclei were isolated from two sources, from the meristematic tip of roots cultured continuously in medium with sucrose and from tips of roots cultured without sucrose. The former had dividing cell randomly distributed in the cell cycle while the latter has little to no mitosis and the meristematic cells are accumulated in the G1 and G2 phases of the cell cycle (24, 25). These two sources represent different physiological conditions and offer a control for the character of the extracted exDNA molecules.
Isolation of exDNA fragments Batches of 25 roots, from a total of 900 roots, were washed with ice-cold sodium phosphate buffer (0.132 M, pH 6.8), treated for 30 sec with 2°7o formaldehyde in phosphate buffer, washed three times with liberal amounts of ice-cold buffer and the 3 mm meristematic tip cut from the root. Each tip, placed on a subbed microscope slide (slides coated with 0.507o gelatin - 0.0507o Crk(SO4)2 • 12H20 and air dried) in about 25/A of buffer, was covered with another slide and tightly pinched between the thumb and index finger, forcing a drop of nuclei at the slide's edge. Drops of nuclear suspension from 25 root tips collected in a chilled 1.5 ml Eppendorf tube were centrifuged at 3 0 0 x g for 5 min at 4 °C to pellet the nuclei. After removal of the supernatant, 100/~1 of selfdigested pronase (10 mg/ml) were added to each pellet and the mixture incubated at room temperature for 1 h. The nuclei were lysed and the
DNA fragments extracted as described by Hirt (11). This involved the careful addition of 150/~1 of 1.2°70 SDS, 0.02 M EDTA, pH 7.5, gently bringing the tube to a horizontal position once and allowing lysis to occur at room temperature for 20 min. The solution adjusted to 1.3 M NaC1 by addition of 75/zl of 5 M NaC1 was mixed by gently rocking the tube at a horizontal position 10 times and placed in ice in a cold room (4 °C) for at least 8 h. The precipitated material was pelleted by centrifugation in the cold at about 13000xg for 15 min in a Beckman microfuge, the tubes removed and flicked with the index finger to dislodge any precipitate attached to the side of the tubes, centrifuged again for 15 min, and placed in ice for 30 min to harden the pellet. The supernatants were combined in a polyallomer tube and the DNA pelleted by centrifugation in a Beckman SW 50.1 rotor at 50000 rpm at 2 °C for 150 min. The DNA was resuspended by vortexing and the solution transferred to a 1.5 ml Eppendorf tube. The polyallomer tube was washed with an additional 100/xl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), vortexed and the wash added to the DNA solution. The DNA was incubated with pancreatic RNase (10/zl of 1 mg/ml RNase per 100/zl of solution) for 1 hr at 37 °C, the solution was extracted with an equal volume of TE-saturated phenol, and the DNA precipitated by the addition of 2 volumes of ice-cold 95070 ethanol and storing at - 2 0 ° C overnight. The precipitate was pelleted by centrifugation in a Beckman microfuge at 13000xg for 30 min, the supernatant removed, the pellet washed with 67070 ethanol chilled to - 2 0 ° C , allowed to stand in ince for 30 min, and again spun at 13000xg for 15 rain. The pellet was dried by lyophilization, dissolved in 1/4 strength TE buffer and dialyzed on a Millipore VSWP filter for 1 h at 22°C against 1/4 strength TE and lyophilized.
Separation of methylated exDNA fragments ExDNA was digested with the restriction endonuclease Hpa II and the fragments separated by gel electrophoresis in a low melting temperature agarose. Fragments larger than 4.3 kb were recovered from the stained gel by melting and phenol extraction (19).
355
Extraction of total D N A The method of Burr and Burr (4) was used to extract total D N A from cultured root tips grown in medium with sucrose for 3 days.
stained gels were exposed to 2 min of 254 nm UV light. After transfer the gels were stained and checked to assure the transfer was complete. The radioactivity applied to each blot ranged from 2 . 2 - 3 . 0 x 1 0 6 cpm and exposure to x-ray film varied from 6 to 48 h depending on the experiment.
Isolation of mitochondrial and plastid DNA The method of Kolodner and Tewari (15, 16, 17) was used to obtain organelle D N A from roots cultured with sucrose for 3 days. The procedure was scaled downward to accommodate small amounts of tissue.
Pea-cDNA clone The plasmid, pHA-1 a derivative of pACYC184, containing a 9 kb Hind III insert with the 18S, 5.8S, and 25S r R N A genes was a gift from Dr R. E. Cuellar (5). The plasmid was cloned in E. coli HB101 and the insert isolated by published procedures (19).
Results
Isolation of nuclei from cultured root tips When dealing with D N A molecules smaller than chromosomal-size it is necessary to minimize random breakage during the isolation procedure. The best approach is to extract D N A from clean, unbroken nuclei. Our method of isolating nuclei from root tips meets this requirement (Fig. 1). The nuclei seen in Fig. la are from a single 3 m m root tip of
[32P]-labeled probes Mitochondrial DNA, plastid DNA, exDNA, total D N A and r D N A were labeled by nick translation to a specific activity of 2 - 4 x 108 cpm/~g using a kit purchased from Bethesda Research Laboratories.
Restriction enzyme digestion Restriction enzymes obtained from Bethesda Research Laboratories were used according to the supplier's specifications.
Gel electrophores& D N A fragments were separated by electrophoresis in 0.6o70 agarose gels using TAE buffer (0.04 M T r i s . HC1, 0.02 M glacial acetic acid, 1 mM EDTA, p H 8.0).
Southern blot hybridization Fig. 1. Nuclei isolated from the 3-mm tip of pea roots, la, phoD N A transfer to Zeta-Probe (Bio-Rad Laboratories), prehybridization, hybridization, washing and strip-wash conditions followed the method o f Gatti et al. (9). Prior to transfer ethidium bromide
tomicrograph of Feulgen-toluidine blue stained nuclei isolated from a single root tip. Ib, an electronmicrographof pelleted isolated nuclei prepared by Dr M. C. Ledbetter of Brookhaven National Laboratory. The average diameter of round nuclei is 8/~m.
356 pea, and t h o u g h p h o t o g r a p h e d at low magnification, they appear unbroken and free o f cytoplasmic tabs. This is true also for nuclei viewed with the electron microscope as seen in Fig. lb. Such nuclei are a proven source o f unsheared, undegraded chrom o s o m a l D N A . They yield nascent molecules o f 6 to 8 x l 0 s daltons under alkaline conditions (26) and double-stranded molecules that often exceed 1 m m or 2x109 daltons (29). The large size o f these molecules is attributable to gentle isolation o f nuclei and to the inactivation o f nucleases by a short pretreatment with 2°7o buffered formaldehyde (J. B. Schvartzman, personal communication).
Separation of exDNA fragments from organelle DNA O u r m e t h o d o f extracting e x D N A does not exclude some organelle DNAs. To separate e x D N A from organelle D N A s we exploited the fact that organelle D N A has fewer methylated bases than nuclear D N A (2, 3, 10, 22, 23). This difference is detectable in D N A digested by the methyl-sensitive
restriction endonuclease, H p a II. Separation o f the H p a II fragments by gel electrophoresis shows that the enzyme cleaves plastid and mitochondrial D N A s to fragments smaller than 4.3 kb (Fig. 2, lanes 1 and 5, respectively). E x D N A and total D N A , on the other hand, are cleaved to fragments larger than 4.3 kb (Fig. 2, lanes 2 and 3, respectively). Hybridization experiments show that the smaller fragments, present in digested e x D N A but absent in total D N A , are h o m o l o g o u s to organelle D N A . Labeled plastid D N A hybridized to these smaller fragments (Fig. 2a, lane 2) giving a banding pattern identical to that o f digested plastid D N A (Fig. 2b, lane 1). Labeled mitochondrial D N A likewise hybridized to H p a II fragments in the digested e x D N A and these are smaller than 4.3 kb (Fig. 2c, lane 4). There are other points worth noting about this experiment. One concerns the low a m o u n t o f hybridization o f plastid D N A to e x D N A fragments o f approximately 23 kb (Fig. 2a, lane 2). The source o f this light signal is obscure. It may indicate a low level o f h o m o l o g y between some e x D N A
Fig. 2. Ethidium-bromide stained agarose gels of plastid, mitochondrial, exDNA, and nuclear DNA digested with Hpa I1 and autoradiograms of blotted DNAs hybridized to either plastid or mitochondrial DNA probes. Stained gel on the left: lane 1, plastid DNA; lane 2, exDNA; lane 3, nuclear DNA. Approximately 0.1 /~g of DNA was loaded per well. Autoradiogram (a), hybridization of [32p]-plastid DNA to blotted DNAs of the gel on the left; the film was exposed for 30 h. Autoradiogram (b), same as (a) except the film was exposed for 9 h. Stained gel on the right: lane 4, exDNA~ lane 5, mitochondrial DNA. Each lane contained approximately 0.12 ~g of DNA. Autoradiogram (c) hybridization of [32p]-mitocbondrialDNA to blotted DNAs of gel pictured on the right. Film was exposed for 6 h. kb, kilobases, noting sizes of marker DNA.
357 fragments and plastid D N A or it may reflect a small amount of nuclear D N A present in the plastid D N A preparation. Whatever its source, the hybridization is so weak compared to that of fragments less than 4.3 kb that its presence is considered insignificant to the present work. Another point is significant. It concerns the absence of hybridization of either the plastid or mitochondrial D N A probes to total D N A (Fig. 2a, lane 3; data using the mitochondrial probe is not shown). The absence of hybridization under our experimental conditions demonstrates that total D N A is equivalent to nuclear D N A and we shall consider it as such hereafter. Finally, the stained gels show that more than 90°70 of the D N A in the exDNA extract has no homology to either plastid or mitochondrial D N A (Fig. 2, lanes 2 and 4).
Homology o f exDNA to nuclear D N A The lack of hybridization of organelle DNAs to most of the H p a II fragments in exDNA indicates that exDNA is of nuclear origin, but we did not know if exDNA represented a few or many nuclear sequences. Therefore, we labeled exDNA H p a II fragments, freed of organelle DNAs, and probed
six separate digests of nuclear D N A to detect homologous sequences. The range of homology is extensive as seen on the autoradiogram shown in Fig. 3a. The extent of hybridization precluded the detection of any specificity of the exDNA. To see differences between exDNA and nuclear sequences it was necessary to hybridize labeled nuclear D N A to the same blot and compare the pattern of hybridization with that produced with exDNA. The autoradiogram using the nuclear D N A probe is shown in Fig. 3b and it has a different pattern than that produced using the exDNA probe (Fig. 3a). For example, exDNA hybridized less to a 7 kb Bgl 1 fragment (Fig. 3a, lane 4) than did nuclear D N A (Fig. 3b, lane 4). A more exacting demonstration of differences, however, was obtained from densitometric tracings of the autoradiograms (Fig. 4). The tracings, seen as sets of two one superimposed upon the other, represent individual measurement of the first three lanes of the autoradiograms. Figure 4a has tracings of hybridization to nuclear D N A digested with Eco R1 (lane 1 of the autoradiograms). The rough dotted line is that of the exD N A probe, the smooth line, is that of the nuclear D N A probe. The tracings show that exDNA hybridized less than nuclear D N A to 7 and 4.3 kb fragments, and more to fragments of 4.3, 3.3, 1.9 and
Fig. 3. Ethidium-bromide stained agarose gel of nuclear DNA digested with Eco RI, lane 1; Barn HI, lane 2; Hind IlI, lane 3; Bgl I, lane 4; Xho I, lane 5; Hinc II, lane 6. Each lane contained approximately 0.4 p.g of DNA. Autoradiogram (a): hybridization of [32p]_ exDNA, 2.5 × 106 cpm, to blotted DNA. Autoradiogram (b): hybridization of [32p]-nuclear DNA, 2.5 × 106 cpm, to blotted DNA. To achieve approximately the same grain density (a) was exposed 24 h, (b) 48 h. kb, kilobases, noting sizes of marker DNA.
358 e
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I
e
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e.e.
.~
o I
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0
Q e. ~ •
o .
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Fig. 4. Densitometric tracings of each of the first three lanes of the autoradiograms shown in Fig. 2. The rough dotted lines are tracings of grain densities of exDNA hybridized to nuclear DNA. The smooth lines are tracings of nuclear DNA hybridized to nuclear DNA. (a), hybridization to nuclear DNA digested with Eco RI, (b), hybridization to nuclear DNA digested with Bam HI, (c), hybridization to nuclear DNA digested with Hind III. kb, kilobases, noting sizes of marker DNA.
1.5 kb. The second set of tracings measures hybridization to DNA digested with Bam HI (Fig. 4b). It shows that exDNA hybridized less than nuclear DNA to fragments of 10.6 and 4.6 kb, more to those of 5.4 and 3.6 kb and more to six fragments smaller than 3.6 kb. Lastly, Fig. 4c shows tracings of hybridization to DNA digested with Hind III (lane 3 of each autoradiogram). In this case, the most notable difference is the amount of hybridization to a 9.6 kb fragment. Relative to the peak density at 5.8 kb, seen in each autoradiogram, the amount of exDNA that hybridized to the 9.6 kb fragment is 43°70 less than that of the nuclear DNA. E x D N A contains ribosomal genes
Extrachromosomal rDNA occurs frequently in unicellular eukaryotes (18), slime mold (31, 33), and amplified rDNA in oocytes is always extrachromosomal (18). A low level of rDNA amplification is reported also to occur in roots of Allium cepa (1). These observations motivated us to determine if exDNA of pea roots likewise had rRNA se-
quences. For the experiment we used the plasmid pHA-1, a derivative of pACYCI84, as a source of rDNA (5, 14). The 18S, 5.8S, and 25S rRNA genes of pea are located on a single 9 kb Hind III inserted into the plasmid. The isolated, labeled insert was used to probe Hind III digests of nuclear DNA and exDNA from dividing and non-dividing root-tip cells. Figure 5 shows that the probe is homologous to a single 9 kb fragment in the digests of each DNA indicating not only the presence of rDNA sequences on exDNA but also that the sequences are confined to the same fragment as nuclear DNA. The importance of this finding should not be overlooked. It demonstrates that exDNA extracted from cells subjected to different physiological conditions is identical, and that exDNA is not a product of random DNA breakage. We repeatedly detect rDNA sequences on exDNA and they are always on a 9 kb Hind III fragment. This indicates that the rRNA genes are on a contiguous strand of exDNA that is never broken separating the genes. Also, the frequency of rRNA genes in the pea genome is 8× 10 -3 making it improbable on a random basis
359
Fig. 6. Autoradiogram of dot blot of exDNA from dividing
meristems, row (a); exDNA from non-dividing meristems, row (b); nuclear DNA, row (c). The dots werehybridized with cloned [32p]-rDNA. Columns (1), (2), and (3), respectively, 0.1, 0.01, and 0.001 #g of DNA blotted. The film was exposed for 6 h. Fig. 5. Autoradiogram of cloned [32P]-rDNA hybridized to
exDNA from dividing meristems, lane 1; exDNA from nondividing meristems, lane 2; nuclear DNA, lane 3. Each lane contained about 0.1 /zg of DNA digested by Hind II1. The film was exposed for 6 h. Ori, gel origin; kb, kilobases.
that the genes would consistently be extracted as exDNA. Given a genome size of 4.6×10-12 g (20) and 3.9x103 r R N A genes per genome (6, 13), there are about 8 × 107 r R N A genes per 0.1 /~g of pea DNA. E x D N A molecules are approximately 54 kb and 0.1 #g of exDNA represents 1.8 × 109 molecules. I f each molecule has r R N A genes, exDNA would be 400 times richer in these sequences than nuclear DNA. Dot blots of exDNA and nuclear D N A hybridized with labeled cloned rDNA, however, show that exDNA from either dividing or nondividing cells has fewer ribosomal sequences than nuclear D N A (Fig. 6). Scintillation counts of the dot blots with 0.1/zg exDNA were 15 to 25% less than that of nuclear DNA. Assuming that 0.1 /~g of nuclear D N A has 8>(107 r R N A genes, the lower radioactivity hybridized to exDNA corresponding to approximately 6.4 >( 107 genes, 1.7 x 109 less than that expected if each molecule had r R N A sequences. It is unlikely, therefore, that the ribosomal sequences in exDNA are amplified.
Discussion In this paper evidence is presented that exDNA molecules of cultured pea-root cells represent repetitive sequences and r R N A genes in relative
amounts that differ from those found in nuclear DNA. Repetitive sequences, in this instance, is used according to H u m m e l et al. (12) who defined them as sequences given rise to visible bands on a gel after electrophoresis of D N A digested by restriction endonucleases. Unique sequences escaped detection in our experiments because the amounts of D N A used in the Southern blots was 0.4 #g or less. The results of these experiments support our earlier conclusion that exDNA represents certain replicons of late replicating D N A that are excised from the chromosomal duplex of specific cells while they are delayed in late S phase prior to differentiating from G 2 phase (27). Recent work shows that these ceils undergo a pronounced change in nuclear shape during the transition from a meristematic to a differentiated state (28). When finishing replicating their DNA, the cells have spherical or round nuclei, and produce exDNA. U p o n completion of D N A replication, their nuclei become elongated or oblong often reaching lengths five times the diameter of the original sphere. This transformation clearly involves structural changes in the chromosomal duplex and we believe these changes include removal of segments (exDNA) from the chromosomal DNA. Whether or not the transformation also involves amplification of certain sequences, of which r D N A is excluded, remains to be determined. Our attempts, though not exhaustive, to detect homology to transcripts other than r R N A have failed, but the possibility still exists that exDNA has other genes. It is also possible that the production of exDNA in pea-root cells is a phenomenon analogous to
360 that observed in developing cells of Tetrahymena (32) and nematodes (21) where differentiation is associated with chromosomal breakage and gene rearrangements. The production of exDNA likewise is consistent with Varshavsky's idea that extrachromosomal copies of nuclear DNA are produced by 'misfiring' of certain replicons (30). 'Misfiring' refers to the additional replication of replicons above that which occurs normally during each round of DNA replication. If multiple 'misfirings' only involve a single, specific replicon, amplification results, but an additional one or two 'misfirings' in several different replicons would produce extrachromosomal DNA of the variety seen in pea. Judging from the work reviewed by Varshavsky and from that on exDNA in pea, the production of replicon-sized nuclear DNA free of the chromosomal duplex is a mechanism involved in the transition of a cell from a proliferative to a differentiated state.
Acknowledgement Research supported by the U.S. Department of Energy and the Monsanto Agricultural Chemical CO.
9. 10.
11. 12.
13. 14.
15.
16.
17.
18. 19.
20.
21.
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