Chromosoma (Berl.) 52,317 9 by Springer-Verlag 1975
327 (1975)
Circular Ribosomal DNA and Ribosomal DNA: Replication in Somatic Amphibian Cells J e a n - D a v i d R o c h a i x 1, ~, a n d A d r i a n P. Bird 1, ~, 4 1 Department of Biology, Yale University, New Haven, Connecticut 06520, U.S.A., and ~"Institut fiir Molekularbiologie II der UuiversitKt Ziirieh, Z/irich, Switzerland; present adresses: a D6partement de Biologic Mol6culaire, Universit6 de Gen~ve, Suisse, 4 MRC Mammalian Genome Unit, University of Edinburgh, West Mains Road, Scotland
Abstract. Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. - - About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rl~NA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction.
Introduction E l e c t r o n microscopy of rD~NA 1 from p a e h y t e n e stage oocytes has established t h a t ribosomal gene amplification i n Xenopus laevis (Gall, 1968; B r o w n a n d Dawid, 1968) involves a rolling circle i n t e r m e d i a t e (Hourcade et al., 1973 ; R o c h a i x et al., 1974). I n the first p a r t of the present s t u d y we have used similar t e c h n i q u e s to investigate r D N A synthesis i n dividing tissue culture cells. The results allow us to compare the replication of a single fraction of the genome i n two c o n t r a s t i n g cell types: the differentiated, n o n - d i v i d i n g , a m p l i f y i n g oocyte; a n d the r a p i d l y diving fibroblast i n culture. I n the course of this work we n o t i c e d t h a t r D N A p r e p a r a t i o n s from c u l t u r e d cells u n e x p e c t e d l y c o n t a i n e d a low p r o p o r t i o n of circular molecules. The second half of the paper describes some properties of these circles. The available evidence suggests t h a t the circles are rDNA, t h a t t h e y are n o t artefacts formed after e x t r a c t i o n of the DNA, a n d t h a t t h e y are able to replicate as circles.
E x p e r i m e n t a l Procedures
DNAs. Six culture flasks of Xenopus laevis kidney cells were grown in the dark in Eagle's Minimum medium supplemented with lactalbumin hydrolysate and foetal calf serum. About one day before confluent growth was achieved, 100 ~Ci of aH-methyl-thymidine (48.5 Ci/mM) and 100 ~Ci of 3H-deoxycytidine (20 Ci/mM) were added for 2 hours. The cells were recovered from the dishes with 3 ml Viokase (Grand Island Biological Company). Five ml of 0.5 M EDTA, 10 mM Tris, pH 9.5, 2% sarkosyl, prewarmed to 60~ C, were added and the mixture was incubated at 60~ C for 1/2 hour. Two ml of pronase (10 mg/ml; predigested for 2 hours at 37~ C) were added and the incubation was continued at 50~ C for 4 to 5 hours. The lysate was ex1 Abbreviations used: rDNA, ribosomal DNA, fraction of the nuclear DNA which contains the sequences of the 18S and 28S ribosomal RNA genes, rRNA, 18S and 28S ribosomal I~NA. SSC, 0.15 M NaC1, 0.015 M ~ a citrate.
318
J.-D. Rochaix and A. P. Bird CPM
300
200
100
0
l
I
IO FRACTION
I
2~0 NUMBER
Fig. 1. Third CsC1 density centrifugation of culture cell ~H-rDNA. The bottom of the gradient is on the left. 20 ~l from each fraction were precipitated with TCA in glass fiber filters. The filters were dried and counted. Fractions 10 to 14 (between the arrows) were pooled and used for the experiments
tracted with distilled phenol saturated with 0.1 M Na borate overnight in the cold. The aqueous phase was precipitated with two volumes of 95% ethanol, the DNA fibers spooled on a glass rod, rinsed in 70% ethanol, resuspended in 0.1 • SSC, mixed with CsC1 to a final density of 1.71 g/cc and spun at 35,000 rev./min in an A1 50 Beckman rotor for 72 hours. The ribosomal DNA containing fractions of the gradients were pooled and precipitated overnight at 0 ~ C with 3 volumes of 70% ethanol. The precipitate was resuspended in 0.1 • SSC and incubated at 37 ~ C for 45 minutes with 40 ~g/ml pancreatic RNase and 20 U/ml T1 l~NAse. The ribosomal DNA was purified further by two consecutive CsC1 density gradient centrffugations. Fig. 1 shows the radioactivity profile of the last gradient. The specific activity of the DNA was 27,200 cpm/~g. Blood rDNA was prepared by equilibrium centrifugation of total DNA in one or two actinomycin/CsC1 gradient (Brown et al., 1971) followed by two CsC1 density gradients. Ribosomal DNA was localized by hybridization with Xenopus ~H-rRNA. Some of the blood rDNA used in this study was kindly donated by Dr. Ken Gross. 3H-rRNA was a gift from Dr. J i m Speirs. Electron Microscope Autoradiography. DNA was spread as described by Davis et al. (1971) except that the cation concentration was 0.025 ~ in the spreading solution and 0.001 M in the hypophase. The size of the culture cell rDNA molecules was measured as described previously (Rochaix et al., 1974). Blood rDNA was scanned in the absence of marker circles, but was sized against a diffraction grating which had been calibrated with PM2 DNA (molecular weight: 6.3 • 106 daltons). P~[2 DNA was a generous gift from Dr. P. Borst. The technique of electron microscope autoradiography has been described (Rochaix et al., 1974). Results and Discussion
RiboSomal D N A Replication Ribosomal DNA was purified ~rom Xenopus cultured cells after a 2 hour labelling with att-thymidine and 3H-deoxycytidine. The purified rDNA 1 was spread for electron microscopy and coated with radiosensitive emulsion for autoradiography. After exposure for two months,
Ribosomal DNA in Somatic Amphibian Cells
319
Table 1. Frequencies of labelled DNA molecules
(~)
(b)
(c)
Lincars Forks Circles Tailed circles Eye forms
92.9 (866) 5.9 (55) 0.7 (6) 0.2 (2) 0.3 (3)
92.0 (167) 7.0 (2) 0.5 (1) 0.5 (1) 0
70.0 6.0 6.0 18.0 0
Total
100 (932)
100 (181)
(47) (4) (4) (12)
100 (67)
The numbers in parenthesis refer to the number of molecules scored. Only molecules carrying at least two or more silver grains were counted for column (a). Column (a), 2 hour labelled culture somatic cell rDNA; column (b), 6 hour labelled ovarian chromosomal DNA; column (c), 0.5-2 hour labelled ovarian amplified rDNA. Columns (b) and (c) are taken from Rochaix et al. (1974). developed autoradiographs were scanned for molecules associated with silver grains, since these must contain DNA synthesized during the 2 hour pulse (Rochaix et al., 1974). The structure of each labelled molecule was recorded in order to determine the frequency of any replication intermediates. The result are presented in Table I a. Most labelled culture cell r D N A molecules are linear and this is p r o b a b l y due to breakage of replication intermediates during handling (Rochaix et al., 1974). B y far the most frequent nonlinear structures are molecules containing a single fork. Moreover, 3 labelled " e y e - f o r m s " (Schn6s and I n m a n , 1970) were identified. Fig. 2 shows one of them. I n all three the requirement t h a t the lengths of the D N A strands connecting the 2 forks should be equal was met. B o t h forks and eye-forms are the expected structures if r D N A replication in cultured ceils follows the p a t t e r n established for bulk chromosomal D N A replication during S phase ( H u b e r m a n and Riggs, 1968 ; Callan, 1972 ; Blumenthal et al., 1973). This view is supported when Table l a is compared with equivalent autoradiographic data for ovarian chromosomal D N A (Table l b). The resemblance suggests t h a t r D N A replication in culture is proceeding b y a mechanism similar to t h a t of bulk ovarian chromosomal DNA. On the other hand, comparison of the frequencies of labelled molecules from culture cell r D N A and from amplifying oocyte r D N A (Table 1 c) shows t h a t r D N A replication during S phase differs from r D N A replication during amplification. The most frequent replication structures in amplified r D N A are rolling circles rather t h a n forks, and no eye-form were seen. Circular Molecules
I n addition to the linear and forked molecules discussed above, labelled r D N A from cultured cells also showed 6 circles and 2 tailed circles. Fig. 3 shows one labelled circular molecule. Since circles were not expected in the r D N A fraction, further observations were made to test whether t h e y were once present in the living cell. W h e n r D N A spreads which had not been autoradiographed were scanned extensively, a p p r o x i m a t e l y 0.15% of the molecules were circular7. Of 56 circles seen, 10 had tails, and 4 were arranged in two pairs of interlocking circles~ one of which is shown in Fig. 4. I n one pair the interlocked rings were each
320
J.-D. Rochaix and A. P. Bird
Fig. 21. Autoradiograph of a replicating rDNA molecule. Kidney culture cells were pulse labelled for 2 hours and the rDNA prepared for electron microscope autoradiography as described in Experimental Procedures. The silver grains appear as large worm-like structures. The two forks are indicated b y arrows. The two branches of the " e y e " have the same size, 18.9 • 106 daltons. The significance of the short single stranded DNA lying adjacent to one b r a n c h of the eye is not known (none was seen on the other eye-forms) 1 The scales on the photos correspond to 0.5 ~.
Ribosomal DNA in Somatic Amphibian Cells
321
Fig. 3. Labelled circular molecule in rDNA. The rDNA was prepared as described in Fig. 2 from culture cells. The sma.ll circles are sX I~F molecules used as internal size markers. The molecular weight of the circle is 30.9 • 106 daltons which corresponds to 4 rDNA units in length
r • 106 daltons and in the other pair they were each 74.0 • 108 daltons. The possibility t h a t the latter are not interlocked, but are merely overlapping is highly unlikely for two reasons. Firstly, the extreme rarity of circles makes overlapping on the grid are very improbable event ; and secondly, despite the rather wide range of circle sizes, the molecular weights within each pair of rings are equal. |~ Are the circles rDNA ? Firstly, t h e y are only found at the buoyant density of rDNA; scanning over 3,000 molecules from the main chromosomal DNA failed to show a single circle. Secondly, it is evident from Figs. 5 and 6 a that although circles displayed a variety of contour lengths ranging up to 90 • 108 daltons (Fig. 5), none smaller than one rDNA repeat unit (7.5 ~ 0.5 • 108 daltons) were seen, and the smallest circles were Blustered at unit length (Fig. 6 a). Both these observations suggest that the circles are rDNA. Partial denaturation, another criterium for rDNA identification (Hourcade et al., 1973), was not possible because the low proportion of existing circles suitably denatured and spread by this procedure made scanning impractical. I t is formally possible that circular molecules in a repeated DNA such as rDNA m a y arise after extraction b y fortuitous base pairing of complementary single stranded ends. The fact that two pairs of equal sized interlocking rings (Fig. 4) were found argues strongly against this possibility since an in vitro origin for such paired oligomers of the rDNA unit is highly unlikely. I n addition a single supereoiled circle, two rDNA units in length, was seen and is shown in Fig. 7. A eovalently closed ring could not have originated by simple base pairing.
322
J.-D. Rochaix and A. P. Bird
Fig. 4. Interlocked circles in rDNA. The rDNA was prepared from cultured cells as described in Fig. 2. The two circular molecules have the same molecular weight, 41.2 • 6 daltons. V refers r the interlocked region
Circle Replication The presence of r a d i o a c t i v e circles w i t h i n t h e pulse l a b e l l e d r D N A p r e p a r a t i o n is n o t sufficient to show t h a t circles are t h e m s e l v e s a b l e to r e p l i c a t e , since it is still possible t h a t t h e y are g e n e r a t e d b y c h r o m o s o m a l r D N A d u r i n g or soon a f t e r its replication. H o w e v e r , t h e o b s e r v a t i o n of b o t h i n t e r l o c k e d circles which are equal in size a n d t a i l e d circles suggests t h a t circle r e p l i c a t i o n does i n d e e d occur. The m o s t feasible e x p l a n a t i o n for equal i n t e r l o c k e d circles is t h a t t h e y are caused
Ribosomal DNA in Somatic Amphibian Cells
323
(a) 4
LU C~ LIJ
n-. u_
5
10
15 (b)
5
10
15
CIRCLE LENGTH (rDNA units)
Fig. 5a and b. Frequency distribution of circle and tailed circle sizes in rDNA. (a) Culture cell rDNA circles; (b) culture cell rDNA tailed circles. 3~oleeular size is expressed as multiples of the 7.5 • 106 daltons rDNA repeat unit
i
i .,.
..,-.
..
(a)
(b)
o
L
I
III II 11 4o
MOLECULAR
n 2o
WEIGHT ( x 1() 6 D A L T O N S )
Fig. 6a--c. Size-distribution of circles from rDNA. (a) Culture cell circles; (b) Blood circles; (c) Blood circles spread from 90% formamide. Each vertical line represents one measured circle whose molecular weight is indicated on the abscissa. The thick horizontal lines show the rDNA repeat unit size (7.5 ~ 0.5) and multiples thereof
b y the failure of progeny circles to separate after a r o u n d of replication. This origin has been proposed for equivalent structures in p o l y o m a D N A (Bourgaux, 1973). While recombination could account for interlocked rings, it is most unlikely t h a t in both observed cases the oligomeric circles should be of equal length. Tailed circles m a y be intermediates in circle duplication. T h e y differ from the tailed circles found in amplifying oocytes in t h a t the tails in 19 out of the 22 seen are shorter t h a n the circle circumference (Fig. 8). This observation is consistent with termination of synthesis after one cycle of the replication fork, rather t h a n
324
J.-D. Rochaix and A. P. Bird
Fig. 7. Supercoil in culture cell rDNA. The contour length is equal to 2 rDNA repeat units
2
O0
I
2 TAIl. : ~]RCLE
3
RATIO
Fig. 8. Histogram of tail to circle ratios of culture cell tailed circles
the repeated cycles which are characteristic of rolling circle replication 'Gilbert and Dressier, 1968). The frequency of labelled molecules within~the total population is 9.5 % (e.g., 34 out of 358). Similarly, labelled circles and tailed circles constitute 10.8 % (7 out of 65) of the total population of circles. Hence the circles increase in numbers at approximately the same rate as the other molecules (mostly linears). This implies that the frequency of rings does not undergo sizable fluctuations, but remains constant during cell growth.
Circles in Blood rDNA The data suggest t h a t a small proportion of the rDNA in cultured Xenopus cells is, circular. Since, cells in culture m a y display abnormal DNA synthesis or ~ecombination we have also examined rDNA purified from Xenopus blood cells.
Ribosomal DNA in Somatic Amphibian Cells
325
Circles were again seen at a low frequency: 20 circles among about 40,000 molecules or 0.05 %. All circles were smaller than 40 million daltons and as shown in Fig. 6 b, their sizes appear to form an oligomeric series between 1 and 4 rDNA repeat units. This suggests that they are rDNA circles. As in the case of culture cell circles, the possibility exists that such a low circle frequency in blood rDNA can be attributed to base pairing of fortuitously complementary single stranded ends. Since no interlocked circles or supercoils were seen, we have attempted to check this possibility in other ways. The melting curve of chromosomal rDNA is biphasic with an early transition at 77~ in 0.1 • SSC (Dawid et al., 1970), or 80 ~ C with the salt concentration used in our spreading solution (Kamenetskii, 1971). Blood rDNA was spread in 90% formamide at room temperature. This roughly corresponds to a temperature equal to the early transition (MeConaughy et al., 1969). Under these conditions at least some circles formed by sticky end overlap would be expected to break. However, the observed circle frequency in 90 % Iormamide (5 circles amongst 8,000 molecules, or 0.06 %) was not significantly different from that in 50 % formamidc preparations. Contour lengths again indicate that the circles are rDNA (Fig. 6 c). These results suggest that the circles found in blood rDNA are not in vitro cyelization artefacts.
Signi]icctnce o[ Circles Ribosomal DNA circles have previously been seen in the oocytes of some amphibia (Miller, 1966; Hourcade et al., 1973), dytiscid water beetles (Gall and Rochaix, 1974), and in the macronucleus of Tetr~hymena (Gall, 1974). I n the ooeytes as well as in Tetrahymena (u et al., 1974) the circles are correlated with an amplification of the rDNA level in the nucleus. Circle frequency in cultured cell rDNA is almost two orders of magnitude lower than in germ cell or macronuclear DNA, about 0.15%. Since there are between 450 and 600 copies of the rRNA genes per nucleolar organizer (Birnstiel etal., 1966; Brown and Weber, 1968), and since the size distribution of linear and circular molecules observed in the electron microscope are fortuitously similar, we can calculate that, on average, at least one ribosomal DNA unit is present as a circle per nucleolar organizer. Blood rDNA in our preparations was smaller on average than the culture cell rDNA and showed only 0.05% circles. This percentage converts to one circular rDNA unit for every 3 nuclcolus organizers since the number average molecular weight of linear and circular blood rDNA molecules was similar. Both these estimates are likely to be low because ring breakage during rDNA purification is inevitable. The presence of rDNA circles in somatic cells is of interest because it may bear upon several puzzling phenomena involving the ribosomal I~NA genes. Circles have already been suggested as templates for the inherited increase in ribosomal RNA gene number in Drosophila (l~itossa, 1972). I t is also possible that the independent replication of rDNA during polytenization in Drosophila (Spear and Gall, 1973) involves extrachromosomal rDNA synthesis. Finally, if free rDNA circles are present in all cells throughout development, the triggering event of gene amplification in the germ cells (e.g., of Xenopus) may not be the excision or transcription (Crippa and Toeehini-Valentini, 1971) of the first rDNA copy, but
326
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r a t h e r the selective replication of preexisting circles. One question which r e m a i n s open is whether or n o t free D N A circles (other t h a n m i t o c h o n d r i a l DNA) are l i m i t e d to ribosomal DNA, or whether t h e y also occur with other repeated D N A sequences (e.g., 5S DNA, satellite DNAs).
Acknowledgements. We thank Dr. J. G. Gall for his interest and advice throughout this work. This study was supported by research grants GM 12427 from the National Institute of General Medical Sciences and VC 85A from the American Cancer Society. J.D.R. was supported by a Public Health Service international research fellowship, A.P.B. was a Damon Runyan fellow, and was supported in part by a grant from Swiss Science Foundation N. 38630.72SR to Prof. M. L. Birnstiel. Re~erences Birnstiel, M. L., Wallace, H., Sirlin, J., Fischberg, M.: Localization of the ribosomal DNA complements in the nueleolar organizer region of Xenopus laevis. Nat. Cancer Inst. Monogr. 23, 431~444 (1966) Blumenthal, A. B., Kriegstein, I-L J., Hogness, D. S. : The units of DNA replication in Drosophila melanogaster chromosomes. Cold Spr. Harb. Syrup. quant. Biol. 38, 205-223 (1973) Bourgaux, P. : On the origin of oligomer forms of polyoma DNA. J. molec. Biol. 77, 197-206 (1973) Brown, D.D., Weber, C. S. : Gene linkage by RNA-DNA hybridization I. Unique DNA sequences homologous to 4S RNA, 5S RNA and ribosomal RNA. J. molec. Biol. 34, 661-680 (1968) Brown, D.D., Dawid, I. B. : Specific gene amplification in oocytes. Science 160, 272-280 (1968) Brown, D. D., Wensink, P. C., Jordan, E. : Purification and some characteristics of 5S DNA from Xenopus laevis. Proc. nat. Acad. Sci. (Wash.) 68, 3175-3179 (1971) Callan, H. G. : Replication of DNA in the chromosomes of eukaryotes. Proc. roy. Soc. B 181, 1941 (1972) Crippa, M., Tocchini-Valentini, G. P. : Synthesis of amplified DNA that codes for ribosomal RNA. Proc. nat. Aead. Sci. (Wash.) 68, 2769-2773 (1971) Davis, R., Simon, M., Davidson, N. : Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In: Methods in enzymology, 21D (L. Grossman and K. Moldave, eds.), p. 413-428. New York: Academic Press 1971 Dawid, I. B., Brown, D.D., Reeder, R.: Composition and structure of chromosomal and amplified ribosomal DNAs of Xenopus la~vis. J. molec. Biol. 51, 341-360 (1970) Gall, J. G. : Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. nat. Acad. Sci. (Wash.) 60, 553-560 (1968) Gall, J. G. : Free ribosomal RNA genes in the macronucleus of Tetrahymena. Proc. nat. Acad. Sci. (Wash.) 71, 3078-3081 (1974) Gall, J. G., Rochaix, J . D . : The amplified ribosomal DNA of dy~iscid beetles. Proc. nat. Acad. Sci. (Wash.) 71, 1819-1823 (1974) Gilbert, W., Dressier, D. : DNA replication: The rolling circle model. Cold Spr. Harb. Symp. quant. Biol. 33, 473484 (1968) Hourcade, D., Dressier, D., Wolfson, J. : The amplification of ribosomal RNA genes involves a rolling circle intermediate. Proc. nat. Acad. Sci. (Wash.) 70, 2926-2930 (1973) Huberman, J. A., Riggs, A. D. : On the mechanism of DNA replication in mammalian chromosomes. J. molec. Biol. 32, 327-341 (1968) Kamenetskii, F. : Simplification of the empirical relationship between melting temperature of DNA, its GC content and concentration of sodium ions in solution. Biopolymers 10, 2623-2624 (1971) McConaughy, B., Laird, C., McCarthy, B. : Nucleic acid reassociation in formamide. Biochemistry (Wash.) 8, 3289-3295 (1969) Miller, O. L. : Structure and composition of peripheral nucleoli of salamander oocytes. Nat. Cancer Inst. Monogr. 23, 53-66 (1966)
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Ritossa, F. : Procedure for magnification of lethal deletions of genes for ribosomal P~NA. Nature (Lond.) New Biol. 240, 109-111 (1972) Rochaix, J. D., Bird, A. P., Bakken, A. H. : Ribosomal RNA Gene Amplification by Rolling Circles. J. molec. Biol. 87, 473-487 (1974) SchnSs, M., Inman, R.: Position of branch points in replicating 1 DNA. J. molec. Biol. 51, 61-73 (1970) Spear, B.B., Gall, J. G.: Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster. Proc. nat. Acad. Sci. (Wash.) 70, 1359-1363 (1973) Yao, M. C., Kimmel, A. R., Gorovsky, M. : A Small Number of Cistrons for Ribosomal RNA in the Germinal Nucleus of a Eukaryote, Tetrahymena pyriformis. Proc. nat. Acad. Sci. (Wash.) 71, 3082-3086 (1974) Received August 3, 1975 / Accepted August 13, 1975, by J. G. Gall Ready for press August 21, 1975
327 (1975)
Circular Ribosomal DNA and Ribosomal DNA: Replication in Somatic Amphibian Cells J e a n - D a v i d R o c h a i x 1, ~, a n d A d r i a n P. Bird 1, ~, 4 1 Department of Biology, Yale University, New Haven, Connecticut 06520, U.S.A., and ~"Institut fiir Molekularbiologie II der UuiversitKt Ziirieh, Z/irich, Switzerland; present adresses: a D6partement de Biologic Mol6culaire, Universit6 de Gen~ve, Suisse, 4 MRC Mammalian Genome Unit, University of Edinburgh, West Mains Road, Scotland
Abstract. Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. - - About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rl~NA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction.
Introduction E l e c t r o n microscopy of rD~NA 1 from p a e h y t e n e stage oocytes has established t h a t ribosomal gene amplification i n Xenopus laevis (Gall, 1968; B r o w n a n d Dawid, 1968) involves a rolling circle i n t e r m e d i a t e (Hourcade et al., 1973 ; R o c h a i x et al., 1974). I n the first p a r t of the present s t u d y we have used similar t e c h n i q u e s to investigate r D N A synthesis i n dividing tissue culture cells. The results allow us to compare the replication of a single fraction of the genome i n two c o n t r a s t i n g cell types: the differentiated, n o n - d i v i d i n g , a m p l i f y i n g oocyte; a n d the r a p i d l y diving fibroblast i n culture. I n the course of this work we n o t i c e d t h a t r D N A p r e p a r a t i o n s from c u l t u r e d cells u n e x p e c t e d l y c o n t a i n e d a low p r o p o r t i o n of circular molecules. The second half of the paper describes some properties of these circles. The available evidence suggests t h a t the circles are rDNA, t h a t t h e y are n o t artefacts formed after e x t r a c t i o n of the DNA, a n d t h a t t h e y are able to replicate as circles.
E x p e r i m e n t a l Procedures
DNAs. Six culture flasks of Xenopus laevis kidney cells were grown in the dark in Eagle's Minimum medium supplemented with lactalbumin hydrolysate and foetal calf serum. About one day before confluent growth was achieved, 100 ~Ci of aH-methyl-thymidine (48.5 Ci/mM) and 100 ~Ci of 3H-deoxycytidine (20 Ci/mM) were added for 2 hours. The cells were recovered from the dishes with 3 ml Viokase (Grand Island Biological Company). Five ml of 0.5 M EDTA, 10 mM Tris, pH 9.5, 2% sarkosyl, prewarmed to 60~ C, were added and the mixture was incubated at 60~ C for 1/2 hour. Two ml of pronase (10 mg/ml; predigested for 2 hours at 37~ C) were added and the incubation was continued at 50~ C for 4 to 5 hours. The lysate was ex1 Abbreviations used: rDNA, ribosomal DNA, fraction of the nuclear DNA which contains the sequences of the 18S and 28S ribosomal RNA genes, rRNA, 18S and 28S ribosomal I~NA. SSC, 0.15 M NaC1, 0.015 M ~ a citrate.
318
J.-D. Rochaix and A. P. Bird CPM
300
200
100
0
l
I
IO FRACTION
I
2~0 NUMBER
Fig. 1. Third CsC1 density centrifugation of culture cell ~H-rDNA. The bottom of the gradient is on the left. 20 ~l from each fraction were precipitated with TCA in glass fiber filters. The filters were dried and counted. Fractions 10 to 14 (between the arrows) were pooled and used for the experiments
tracted with distilled phenol saturated with 0.1 M Na borate overnight in the cold. The aqueous phase was precipitated with two volumes of 95% ethanol, the DNA fibers spooled on a glass rod, rinsed in 70% ethanol, resuspended in 0.1 • SSC, mixed with CsC1 to a final density of 1.71 g/cc and spun at 35,000 rev./min in an A1 50 Beckman rotor for 72 hours. The ribosomal DNA containing fractions of the gradients were pooled and precipitated overnight at 0 ~ C with 3 volumes of 70% ethanol. The precipitate was resuspended in 0.1 • SSC and incubated at 37 ~ C for 45 minutes with 40 ~g/ml pancreatic RNase and 20 U/ml T1 l~NAse. The ribosomal DNA was purified further by two consecutive CsC1 density gradient centrffugations. Fig. 1 shows the radioactivity profile of the last gradient. The specific activity of the DNA was 27,200 cpm/~g. Blood rDNA was prepared by equilibrium centrifugation of total DNA in one or two actinomycin/CsC1 gradient (Brown et al., 1971) followed by two CsC1 density gradients. Ribosomal DNA was localized by hybridization with Xenopus ~H-rRNA. Some of the blood rDNA used in this study was kindly donated by Dr. Ken Gross. 3H-rRNA was a gift from Dr. J i m Speirs. Electron Microscope Autoradiography. DNA was spread as described by Davis et al. (1971) except that the cation concentration was 0.025 ~ in the spreading solution and 0.001 M in the hypophase. The size of the culture cell rDNA molecules was measured as described previously (Rochaix et al., 1974). Blood rDNA was scanned in the absence of marker circles, but was sized against a diffraction grating which had been calibrated with PM2 DNA (molecular weight: 6.3 • 106 daltons). P~[2 DNA was a generous gift from Dr. P. Borst. The technique of electron microscope autoradiography has been described (Rochaix et al., 1974). Results and Discussion
RiboSomal D N A Replication Ribosomal DNA was purified ~rom Xenopus cultured cells after a 2 hour labelling with att-thymidine and 3H-deoxycytidine. The purified rDNA 1 was spread for electron microscopy and coated with radiosensitive emulsion for autoradiography. After exposure for two months,
Ribosomal DNA in Somatic Amphibian Cells
319
Table 1. Frequencies of labelled DNA molecules
(~)
(b)
(c)
Lincars Forks Circles Tailed circles Eye forms
92.9 (866) 5.9 (55) 0.7 (6) 0.2 (2) 0.3 (3)
92.0 (167) 7.0 (2) 0.5 (1) 0.5 (1) 0
70.0 6.0 6.0 18.0 0
Total
100 (932)
100 (181)
(47) (4) (4) (12)
100 (67)
The numbers in parenthesis refer to the number of molecules scored. Only molecules carrying at least two or more silver grains were counted for column (a). Column (a), 2 hour labelled culture somatic cell rDNA; column (b), 6 hour labelled ovarian chromosomal DNA; column (c), 0.5-2 hour labelled ovarian amplified rDNA. Columns (b) and (c) are taken from Rochaix et al. (1974). developed autoradiographs were scanned for molecules associated with silver grains, since these must contain DNA synthesized during the 2 hour pulse (Rochaix et al., 1974). The structure of each labelled molecule was recorded in order to determine the frequency of any replication intermediates. The result are presented in Table I a. Most labelled culture cell r D N A molecules are linear and this is p r o b a b l y due to breakage of replication intermediates during handling (Rochaix et al., 1974). B y far the most frequent nonlinear structures are molecules containing a single fork. Moreover, 3 labelled " e y e - f o r m s " (Schn6s and I n m a n , 1970) were identified. Fig. 2 shows one of them. I n all three the requirement t h a t the lengths of the D N A strands connecting the 2 forks should be equal was met. B o t h forks and eye-forms are the expected structures if r D N A replication in cultured ceils follows the p a t t e r n established for bulk chromosomal D N A replication during S phase ( H u b e r m a n and Riggs, 1968 ; Callan, 1972 ; Blumenthal et al., 1973). This view is supported when Table l a is compared with equivalent autoradiographic data for ovarian chromosomal D N A (Table l b). The resemblance suggests t h a t r D N A replication in culture is proceeding b y a mechanism similar to t h a t of bulk ovarian chromosomal DNA. On the other hand, comparison of the frequencies of labelled molecules from culture cell r D N A and from amplifying oocyte r D N A (Table 1 c) shows t h a t r D N A replication during S phase differs from r D N A replication during amplification. The most frequent replication structures in amplified r D N A are rolling circles rather t h a n forks, and no eye-form were seen. Circular Molecules
I n addition to the linear and forked molecules discussed above, labelled r D N A from cultured cells also showed 6 circles and 2 tailed circles. Fig. 3 shows one labelled circular molecule. Since circles were not expected in the r D N A fraction, further observations were made to test whether t h e y were once present in the living cell. W h e n r D N A spreads which had not been autoradiographed were scanned extensively, a p p r o x i m a t e l y 0.15% of the molecules were circular7. Of 56 circles seen, 10 had tails, and 4 were arranged in two pairs of interlocking circles~ one of which is shown in Fig. 4. I n one pair the interlocked rings were each
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Fig. 21. Autoradiograph of a replicating rDNA molecule. Kidney culture cells were pulse labelled for 2 hours and the rDNA prepared for electron microscope autoradiography as described in Experimental Procedures. The silver grains appear as large worm-like structures. The two forks are indicated b y arrows. The two branches of the " e y e " have the same size, 18.9 • 106 daltons. The significance of the short single stranded DNA lying adjacent to one b r a n c h of the eye is not known (none was seen on the other eye-forms) 1 The scales on the photos correspond to 0.5 ~.
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Fig. 3. Labelled circular molecule in rDNA. The rDNA was prepared as described in Fig. 2 from culture cells. The sma.ll circles are sX I~F molecules used as internal size markers. The molecular weight of the circle is 30.9 • 106 daltons which corresponds to 4 rDNA units in length
r • 106 daltons and in the other pair they were each 74.0 • 108 daltons. The possibility t h a t the latter are not interlocked, but are merely overlapping is highly unlikely for two reasons. Firstly, the extreme rarity of circles makes overlapping on the grid are very improbable event ; and secondly, despite the rather wide range of circle sizes, the molecular weights within each pair of rings are equal. |~ Are the circles rDNA ? Firstly, t h e y are only found at the buoyant density of rDNA; scanning over 3,000 molecules from the main chromosomal DNA failed to show a single circle. Secondly, it is evident from Figs. 5 and 6 a that although circles displayed a variety of contour lengths ranging up to 90 • 108 daltons (Fig. 5), none smaller than one rDNA repeat unit (7.5 ~ 0.5 • 108 daltons) were seen, and the smallest circles were Blustered at unit length (Fig. 6 a). Both these observations suggest that the circles are rDNA. Partial denaturation, another criterium for rDNA identification (Hourcade et al., 1973), was not possible because the low proportion of existing circles suitably denatured and spread by this procedure made scanning impractical. I t is formally possible that circular molecules in a repeated DNA such as rDNA m a y arise after extraction b y fortuitous base pairing of complementary single stranded ends. The fact that two pairs of equal sized interlocking rings (Fig. 4) were found argues strongly against this possibility since an in vitro origin for such paired oligomers of the rDNA unit is highly unlikely. I n addition a single supereoiled circle, two rDNA units in length, was seen and is shown in Fig. 7. A eovalently closed ring could not have originated by simple base pairing.
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Fig. 4. Interlocked circles in rDNA. The rDNA was prepared from cultured cells as described in Fig. 2. The two circular molecules have the same molecular weight, 41.2 • 6 daltons. V refers r the interlocked region
Circle Replication The presence of r a d i o a c t i v e circles w i t h i n t h e pulse l a b e l l e d r D N A p r e p a r a t i o n is n o t sufficient to show t h a t circles are t h e m s e l v e s a b l e to r e p l i c a t e , since it is still possible t h a t t h e y are g e n e r a t e d b y c h r o m o s o m a l r D N A d u r i n g or soon a f t e r its replication. H o w e v e r , t h e o b s e r v a t i o n of b o t h i n t e r l o c k e d circles which are equal in size a n d t a i l e d circles suggests t h a t circle r e p l i c a t i o n does i n d e e d occur. The m o s t feasible e x p l a n a t i o n for equal i n t e r l o c k e d circles is t h a t t h e y are caused
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(a) 4
LU C~ LIJ
n-. u_
5
10
15 (b)
5
10
15
CIRCLE LENGTH (rDNA units)
Fig. 5a and b. Frequency distribution of circle and tailed circle sizes in rDNA. (a) Culture cell rDNA circles; (b) culture cell rDNA tailed circles. 3~oleeular size is expressed as multiples of the 7.5 • 106 daltons rDNA repeat unit
i
i .,.
..,-.
..
(a)
(b)
o
L
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III II 11 4o
MOLECULAR
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Fig. 6a--c. Size-distribution of circles from rDNA. (a) Culture cell circles; (b) Blood circles; (c) Blood circles spread from 90% formamide. Each vertical line represents one measured circle whose molecular weight is indicated on the abscissa. The thick horizontal lines show the rDNA repeat unit size (7.5 ~ 0.5) and multiples thereof
b y the failure of progeny circles to separate after a r o u n d of replication. This origin has been proposed for equivalent structures in p o l y o m a D N A (Bourgaux, 1973). While recombination could account for interlocked rings, it is most unlikely t h a t in both observed cases the oligomeric circles should be of equal length. Tailed circles m a y be intermediates in circle duplication. T h e y differ from the tailed circles found in amplifying oocytes in t h a t the tails in 19 out of the 22 seen are shorter t h a n the circle circumference (Fig. 8). This observation is consistent with termination of synthesis after one cycle of the replication fork, rather t h a n
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Fig. 7. Supercoil in culture cell rDNA. The contour length is equal to 2 rDNA repeat units
2
O0
I
2 TAIl. : ~]RCLE
3
RATIO
Fig. 8. Histogram of tail to circle ratios of culture cell tailed circles
the repeated cycles which are characteristic of rolling circle replication 'Gilbert and Dressier, 1968). The frequency of labelled molecules within~the total population is 9.5 % (e.g., 34 out of 358). Similarly, labelled circles and tailed circles constitute 10.8 % (7 out of 65) of the total population of circles. Hence the circles increase in numbers at approximately the same rate as the other molecules (mostly linears). This implies that the frequency of rings does not undergo sizable fluctuations, but remains constant during cell growth.
Circles in Blood rDNA The data suggest t h a t a small proportion of the rDNA in cultured Xenopus cells is, circular. Since, cells in culture m a y display abnormal DNA synthesis or ~ecombination we have also examined rDNA purified from Xenopus blood cells.
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Circles were again seen at a low frequency: 20 circles among about 40,000 molecules or 0.05 %. All circles were smaller than 40 million daltons and as shown in Fig. 6 b, their sizes appear to form an oligomeric series between 1 and 4 rDNA repeat units. This suggests that they are rDNA circles. As in the case of culture cell circles, the possibility exists that such a low circle frequency in blood rDNA can be attributed to base pairing of fortuitously complementary single stranded ends. Since no interlocked circles or supercoils were seen, we have attempted to check this possibility in other ways. The melting curve of chromosomal rDNA is biphasic with an early transition at 77~ in 0.1 • SSC (Dawid et al., 1970), or 80 ~ C with the salt concentration used in our spreading solution (Kamenetskii, 1971). Blood rDNA was spread in 90% formamide at room temperature. This roughly corresponds to a temperature equal to the early transition (MeConaughy et al., 1969). Under these conditions at least some circles formed by sticky end overlap would be expected to break. However, the observed circle frequency in 90 % Iormamide (5 circles amongst 8,000 molecules, or 0.06 %) was not significantly different from that in 50 % formamidc preparations. Contour lengths again indicate that the circles are rDNA (Fig. 6 c). These results suggest that the circles found in blood rDNA are not in vitro cyelization artefacts.
Signi]icctnce o[ Circles Ribosomal DNA circles have previously been seen in the oocytes of some amphibia (Miller, 1966; Hourcade et al., 1973), dytiscid water beetles (Gall and Rochaix, 1974), and in the macronucleus of Tetr~hymena (Gall, 1974). I n the ooeytes as well as in Tetrahymena (u et al., 1974) the circles are correlated with an amplification of the rDNA level in the nucleus. Circle frequency in cultured cell rDNA is almost two orders of magnitude lower than in germ cell or macronuclear DNA, about 0.15%. Since there are between 450 and 600 copies of the rRNA genes per nucleolar organizer (Birnstiel etal., 1966; Brown and Weber, 1968), and since the size distribution of linear and circular molecules observed in the electron microscope are fortuitously similar, we can calculate that, on average, at least one ribosomal DNA unit is present as a circle per nucleolar organizer. Blood rDNA in our preparations was smaller on average than the culture cell rDNA and showed only 0.05% circles. This percentage converts to one circular rDNA unit for every 3 nuclcolus organizers since the number average molecular weight of linear and circular blood rDNA molecules was similar. Both these estimates are likely to be low because ring breakage during rDNA purification is inevitable. The presence of rDNA circles in somatic cells is of interest because it may bear upon several puzzling phenomena involving the ribosomal I~NA genes. Circles have already been suggested as templates for the inherited increase in ribosomal RNA gene number in Drosophila (l~itossa, 1972). I t is also possible that the independent replication of rDNA during polytenization in Drosophila (Spear and Gall, 1973) involves extrachromosomal rDNA synthesis. Finally, if free rDNA circles are present in all cells throughout development, the triggering event of gene amplification in the germ cells (e.g., of Xenopus) may not be the excision or transcription (Crippa and Toeehini-Valentini, 1971) of the first rDNA copy, but
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r a t h e r the selective replication of preexisting circles. One question which r e m a i n s open is whether or n o t free D N A circles (other t h a n m i t o c h o n d r i a l DNA) are l i m i t e d to ribosomal DNA, or whether t h e y also occur with other repeated D N A sequences (e.g., 5S DNA, satellite DNAs).
Acknowledgements. We thank Dr. J. G. Gall for his interest and advice throughout this work. This study was supported by research grants GM 12427 from the National Institute of General Medical Sciences and VC 85A from the American Cancer Society. J.D.R. was supported by a Public Health Service international research fellowship, A.P.B. was a Damon Runyan fellow, and was supported in part by a grant from Swiss Science Foundation N. 38630.72SR to Prof. M. L. Birnstiel. Re~erences Birnstiel, M. L., Wallace, H., Sirlin, J., Fischberg, M.: Localization of the ribosomal DNA complements in the nueleolar organizer region of Xenopus laevis. Nat. Cancer Inst. Monogr. 23, 431~444 (1966) Blumenthal, A. B., Kriegstein, I-L J., Hogness, D. S. : The units of DNA replication in Drosophila melanogaster chromosomes. Cold Spr. Harb. Syrup. quant. Biol. 38, 205-223 (1973) Bourgaux, P. : On the origin of oligomer forms of polyoma DNA. J. molec. Biol. 77, 197-206 (1973) Brown, D.D., Weber, C. S. : Gene linkage by RNA-DNA hybridization I. Unique DNA sequences homologous to 4S RNA, 5S RNA and ribosomal RNA. J. molec. Biol. 34, 661-680 (1968) Brown, D.D., Dawid, I. B. : Specific gene amplification in oocytes. Science 160, 272-280 (1968) Brown, D. D., Wensink, P. C., Jordan, E. : Purification and some characteristics of 5S DNA from Xenopus laevis. Proc. nat. Acad. Sci. (Wash.) 68, 3175-3179 (1971) Callan, H. G. : Replication of DNA in the chromosomes of eukaryotes. Proc. roy. Soc. B 181, 1941 (1972) Crippa, M., Tocchini-Valentini, G. P. : Synthesis of amplified DNA that codes for ribosomal RNA. Proc. nat. Aead. Sci. (Wash.) 68, 2769-2773 (1971) Davis, R., Simon, M., Davidson, N. : Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In: Methods in enzymology, 21D (L. Grossman and K. Moldave, eds.), p. 413-428. New York: Academic Press 1971 Dawid, I. B., Brown, D.D., Reeder, R.: Composition and structure of chromosomal and amplified ribosomal DNAs of Xenopus la~vis. J. molec. Biol. 51, 341-360 (1970) Gall, J. G. : Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. nat. Acad. Sci. (Wash.) 60, 553-560 (1968) Gall, J. G. : Free ribosomal RNA genes in the macronucleus of Tetrahymena. Proc. nat. Acad. Sci. (Wash.) 71, 3078-3081 (1974) Gall, J. G., Rochaix, J . D . : The amplified ribosomal DNA of dy~iscid beetles. Proc. nat. Acad. Sci. (Wash.) 71, 1819-1823 (1974) Gilbert, W., Dressier, D. : DNA replication: The rolling circle model. Cold Spr. Harb. Symp. quant. Biol. 33, 473484 (1968) Hourcade, D., Dressier, D., Wolfson, J. : The amplification of ribosomal RNA genes involves a rolling circle intermediate. Proc. nat. Acad. Sci. (Wash.) 70, 2926-2930 (1973) Huberman, J. A., Riggs, A. D. : On the mechanism of DNA replication in mammalian chromosomes. J. molec. Biol. 32, 327-341 (1968) Kamenetskii, F. : Simplification of the empirical relationship between melting temperature of DNA, its GC content and concentration of sodium ions in solution. Biopolymers 10, 2623-2624 (1971) McConaughy, B., Laird, C., McCarthy, B. : Nucleic acid reassociation in formamide. Biochemistry (Wash.) 8, 3289-3295 (1969) Miller, O. L. : Structure and composition of peripheral nucleoli of salamander oocytes. Nat. Cancer Inst. Monogr. 23, 53-66 (1966)
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Ritossa, F. : Procedure for magnification of lethal deletions of genes for ribosomal P~NA. Nature (Lond.) New Biol. 240, 109-111 (1972) Rochaix, J. D., Bird, A. P., Bakken, A. H. : Ribosomal RNA Gene Amplification by Rolling Circles. J. molec. Biol. 87, 473-487 (1974) SchnSs, M., Inman, R.: Position of branch points in replicating 1 DNA. J. molec. Biol. 51, 61-73 (1970) Spear, B.B., Gall, J. G.: Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster. Proc. nat. Acad. Sci. (Wash.) 70, 1359-1363 (1973) Yao, M. C., Kimmel, A. R., Gorovsky, M. : A Small Number of Cistrons for Ribosomal RNA in the Germinal Nucleus of a Eukaryote, Tetrahymena pyriformis. Proc. nat. Acad. Sci. (Wash.) 71, 3082-3086 (1974) Received August 3, 1975 / Accepted August 13, 1975, by J. G. Gall Ready for press August 21, 1975
