Preferential replication of the ribosomal RNA genes during a nutritional shift-up in Tetrahymena pyriformis

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BIOCHIM1CA ET BIOPHYSICA .\('IA

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P R E F E R E N T I A L R E P L I C A T I O N OF T H E RIBOSOMAL RNA G E N E S D U R I N G A N U T R I T I O N A L S H I F T - U P IN TETRAHYMENA PYRIFORMIS J A N E N G B E R G * , D A V I D M O W A T AND R O N A L D E. P E A R L M A N

Department of Biology, York University, Toronto, Ontario (Canada) (Received J a n u a r y 24th, 1972)

SUMMARY

A preferential initiation of replication of rRNA genes in Tetrahymena is induced when starved cells are shifted to nutrient medium. This preferential replication of r R N A genes suggests that initiation of replication of bulk nuclear DNA and nucleolar DNA are under separate controls. Preferential replication of r R N A genes does not result in gene amplification in this system.

INTRODUCTION

Cytological observations in the ciliate protozoan Tetrahymenapyri]ormis have demonstrated changes in nucleolar organization and substructure in response to changes in the cellular environment 1-5. The multiple peripherally located small nucleoli in the macronuclei of exponentially growing Tetrahymena aggregate to a few large fusion bodies when the cells are starved by transfer to an inorganic medium. Refeeding of the cells with fresh growth medium induces dissociation of the fusion bodies into small nucleoli and the typical log phase nucleolar organization is attained within 2-3 h of refeeding s. It is not apparent from cytological observations whether changes in nucleolar organization and total surface area are associated with changes in total nucleolar mass or nucleolar DNA. Nucleolar DNA in Tetrahymenahas recently been shown to include the r R N A genes (J. Engberg, unpublished observations) in agreement with observations in other organisms s. As it is known that the synthesis of RNA begins at a high rate 2-3 h after refeeding of starved cells 5 we wanted to test if the change in net RNA synthesis could be related to a change in the amount of rRNA genes in the cell. By using the technique of D N A - R N A hybridization we have recently shown that the amount of rRNA genes is 30 % more in exponentially growing cells than in starved cells 7. In the study reported here, cells were starved and then refed. DNA was isolated and the DNA synthesized during the early part of the refeeding period was found to be enriched with respect to the rRNA genes indicating that a preferential replication of these genes takes place in response to the nutritional shift-up. A b b r e v i a t i o n s : BrdU, 5 - b r o m o d e o x y u r i d i n e ; SSC, O.Ol 5 ~V[ s o d i u m c i t r a t e - o . I 5 M N a ( l (pH 7.0). * P r e s e n t address: Biochemical I n s t i t u t e ]3, J u l i a n n e Maries Vej 3 o2, Copenhagen D K 21oo, Denmark.

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MATERIALS AND METHODS

Cultures of Tetrahymena pyri]ormis, amicronucleate strain GL were grown with vigorous aeration at 28°C in a defined medium s supplemented with 0.5 % proteose peptone (Difco). Cell density was determined electronically using a Coulter counter Model B. Starvation was accomplished by transfer of exponentially growing cells (generation time 3 h) to inorganic medium and further incubation for 24 h as described by Cameron and Jeter 9. Nuclei from exponentially growing and starved cells were prepared by the method of Lee and Byfield1° except that the nuclei were pelleted through 25 % Ficoll instead of through 0. 5 M sucrose:. Nuclei were prepared for electron microscopy as described by Nilsson and Leick 5. DNA in exponentially growing Tetrahymena was uniformly labeled by growth for 8 generations in o.I/xCi/ml [aH]thymidine (New England Nuclear Corp., specific activity io Ci/mmole). DNA from nuclei was prepared and analysed in CsC1 density gradients by the method of Brunk and Hanawalt n as described previously:. Each gradient consisted of 7.2 g CsC1 and 6.0 ml buffer containing about 5o/tg DNA. Centrifugation was at 37 ooo rev./min for 60 h at 22°C in the A-32I fixed angle rotor of an International B-6o ultracentrifuge. About 4 ° fractions of equal volume were collected through the bottom of the centrifuge tube (Polyallomer, Beckman). Buoyant density determinations were made using a Zeiss Abbe refractometer (Model A) as described previously12. The observed values were normalized using E. coli B DNA with buoyant density 1.71o g/ml as reference. For preparation of purified 3*P-labeled rRNA, cultures of Tetrahymena were grown in defined medium s without phosphate, supplemented with 0.25 % proteose peptone. The medium contained 50 ktCi/ml of 32P1 (carrier free, New England Nuclear Corp.). Ribosomes were pelleted by centrifugation 12. RNA was obtained from the ribosomes by phenol extraction as described by Leick and Plesner 13. The RNA was analysed on 5-2o % sucrose gradients in IO mM Tris-HC1 buffer (pH 7.4), I mM EDTA, 50 mM NaC1 and 0.25 % sodium dodecyl sulfate. Peak fractions of the 25-S and I7-S rRNA were pooled and precipitated with ethanol. The 25-S and I7-S rRNA was further purified on two successive sucrose gradients and finally passed through membrane filters as described by Nygaard and Hall 14. The 3~P-labeled rRNA had an initial specific activity of 12o ooo cpm/ktg. An equimolar mixture of 25-S and I7-S rRNA was used for the hybridization experiments. Exponentially growing cells were pulse labeled for 5 min with o.I mCi/ml 8*Pt (carrier free, New England Nuclear Corp.). Total nucleic acids were extracted by the modified diethyl procarbonate Method II of Solymosy et al. 15 and subsequently treated with 20 ktg/ml deoxyribonuclease (Worthington, electrophoretically pure) for I h at 37°C in Tris-Mg 2+ buffer le. After several phenol extractions and ethanol precipitations the RNA solution was further purified by passing it twice through membrane filters 1.. For DNA-RNA hybridization, DNA from density gradient fractions was denatured in alkali and loaded on separate cellulose nitrate filters (Sartorius, G6ttingen, West Germany) by the method of Birnstiel et al. 1:. The loading efficiency of DNA was greater than 95 % based on acid precipitable radioactivity. Each DNA loaded filter was incubated with s*P-labeled RNA in I ml I × SSC (O.Ol5 M sodium citrate-o.I5 M NaC1 (pH 7.o)), 50 % formamide at 37°C for 24 h. After incubation the filters were Biochim. Biophys. Acta, 272 (I972) 312-32o

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processed b y the m e t h o d of Gillespie a n d Spiegellnan TM as described elsewlL~'rc:. Samt)les were c o u n t e d in a 13eckman LS e33 scintillation counter using t,,hwm~ scintillator fluid.

RESULTS

Fig. I shows t h a t tile changes in tile i n t r a n u c l e a r localization of tile nucleoli p r e v i o u s l y o b s e r v e d in whole cells 5 are p r e s e r v e d when nuclei are isolated from t h e cells. N u m e r o u s small p e r i p h e r a l l y located nucleoli are o b s e r v e d in nuclei is(~lated

A

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Fig. I. Electron m i c r o g r a p h s of cross sections t h r o u g h m a c r o n u c l e i isolated f r o m e x p o n e n t i a l l y g r o w i n g /A) a n d s t a r v e d (B) Tetrahymena pyriformis G L (amicronucleate). C u l t u r e s of Tetrahymena were g r o w n a n d nuclei were isolated as described in Materials a n d l~ethods. A. N u m e r o u s small, cup s h a p e d nucleoli (N') are d i s t r i b u t e d along t h e i n n e r n u c l e a r m e m b r a n e (Y[): c h r o m a t i n g r a n u l e s (C). × 23 2oo. /3. A few large nucleolar a g g r e g a t e s or fusion bodies (FB) a p p e a r as a r e s u l t of s t a r v a t i o n . G r a n u l a r nucleolonema-like e l e m e n t s (NM) are observed. T h e s e e l e m e n t s are n e v e r seen in e x p o n e n t i a l l y g r o w i n g cells s. × 43 200.

from exponentially growing Tetrahymena.As a result of starvation, a few large nucleolar aggregates or fusion bodies appear in isolated nuclei. This observation together with the demonstration that the amount of rRNA genes is 30 % more in exponentially Biochim. Biophys. Acta, 272 ( 1 9 7 2 ) 3 1 2 - 3 2 o

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growing cells than in starved cells suggests that replication of rRNA genes and the rest of the nuclear DNA are under separate controls 7. To test this hypothesis, we used the techniques of D N A - R N A hybridization and CsC1 density gradient centrifugation to ask whether selective replication of rRNA genes could be observed under certain physiological conditions.

Buoyant density o/the rRNA genes Since the rRNA genes (GC = 43 % (ref. 18)) have a different GC content than the bulk DNA (GC --~ 29 % (ref. 19) ) they are expected to band at a different position than the bulk DNA in a CsC1 density gradient 2°. The banding position of the rRNA genes was determined by challenging every fraction of a CsC1 gradient of DNA with 32P-labeled r R N A as can be seen in Fig. 2a. As expected, the hybridization curve (32p) which represents the banding position of the rRNA genes, has a higher buoyant density than the bulk DNA (all). The specificity of the hybridization reaction is described elsewhere 7. The buoyant density of the r R N A genes (1.694 g/ml) is identical to a small DNA satellite in Tetrahymena observed in analytical CsC1 density gradient centrifugation b y Flavell and Jones ~1 and Suyama ~2. When DNA fractionated on a CsC1 density gradient is challenged with pulselabeled total cellular RNA (Fig. 2b), the hybridization curve parallels the DNA curve

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Fig. 2. B u o y a n t d e n s i t y of t h e rR2qA genes in CsCI d e n s i t y gradients. D N A isolated f r o m nuclei f r o m u n i f o r m l y labeled e x p o n e n t i a l l y g r o w i n g Tetrahymena w a s p r e p a r e d a n d a n a l y s e d in CsC1 d e n s i t y g r a d i e n t s as described in M a t e r i a l s a n d M e t h o d s . D l g A f r o m t h e d e n s i t y g r a d i e n t fract i o n s w a s loaded on s e p a r a t e nitrocellulose filters for D N A - R N A h y b r i d i z a t i o n . A. E a c h D N A loaded filter w a s i n c u b a t e d w i t h 2 fig purified s2P-labeled r R N A as described in Materials a n d M e t h o d s . B i n d i n g to b l a n k filters (no D N A ) a n d m a c h i n e b a c k g r o u n d h a v e been s u b t r a c t e d f r o m d a t a p r e s e n t e d . B. E a c h D l g A loaded filter w a s i n c u b a t e d w i t h i o o / ~ g p u l s e labeled t o t a l cellular I~:NN as described in Materials a n d M e t h o d s . B i n d i n g to b l a n k filters (no DlgA) a n d m a c h i n e b a c k g r o u n d h a v e been s u b t r a c t e d f r o m d a t a p r e s e n t e d .

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(1.686 g/ml) with no sign of a shoulder in the DNA satellite position (1.694 g/ml). This indicates that the rRNA used in Fig. 2a was free of contamination with shortlived mRNAs and that hybridization with the DNA in the satellite position represents rRNA-DNA hybrids under the conditions described.

Test o/selective DNA replication Because of the small difference in buoyant density between the rRNA genes and the bulk DNA in CsC1 density gradient analysis, it was not possible to visualize a preferential synthesis of the rRNA genes by CsC1 density gradient analysis as has been used by Brown and Dawid~ and Gall** in their study of amphibian oocytes. In order to test for a selective replication of the rRNA genes during some parts of the refeeding period, the DNA replicated during selected intervals of the refeeding period was separated from the remainder of the nuclear DNA and subsequently tested for its ability to hybridize with rRNA. The isolation of the DNA replicated during selected intervals of the refeeding period was accomplished by refeeding starved cells with growth medium containing 5-bromodeoxyuridine (BrdU), which is utilized in place of thymidine in DNA replicationxx, and separating the BrdU substituted DNA from non-replicated DNA by CsC1 density gradient centrifugation*s. In the experiment depicted in Fig. 3, a culture of Tetrahymena was prelabeled with [3H]thymidine before transfer to starvation medium. The arrows (Fig. 3) indicate the position where newly synthesized DNA containing BrdU is known to band. After 9° min of refeeding,

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Fig. 3. K i n e t i c s of t h e replication of t h e ' r R B I A genes a f t e r refeeding of s t a r v e d cells. E x p o n e n t i a l ly g r o w i n g cells were prelabeled w i t h [sI-~]thymidine (o. 5 /~Ci/ml) a n d t r a n s f e r r e d to inorganic m e d i u m °. A f t e r 24 h of s t a r v a t i o n , i vol. of 2 t i m e s c o n c e n t r a t e d g r o w t h m e d i u m w a s a d d e d t o g e t h e r w i t h B r d U a t a final c o n c e n t r a t i o n of 50/~g/ml. Cell s a m p l e s were t a k e n a t 90, 15o a n d 270 m i n after refeeding a n d D • A w a s a n a l y s e d on CsC1 d e n s i t y g r a d i e n t s (7.4 g CsC1 a n d 6.o ml buffer) a n d loaded on m e m b r a n e filters as described in Materials a n d M e t h o d s . E a c h D N A loaded filter w a s i n c u b a t e d w i t h 2 /~g/ml purified *2P-labeled rRBIA a n d processed as described in Materials a n d M e t h o d s . I n a parallel e x p e r i m e n t (not s h o w n ) ssP t w a s included in t h e refeeding m e d i u m in order to a s c e r t a i n t h e b a n d i n g position of t h e n e w l y s y n t h e s i z e d B r d U c o n t a i n i n g D N A in t h e CsC1 d e n s i t y g r a d i e n t s u s e d in t h e s e e x p e r i m e n t s . T h i s p o s i t i o n is i n d i c a t e d w i t h a n a r r o w in t h e g r a p h s . T h e p r o p o r t i o n of t h e t o t a l a m o u n t s of sI-~ c o u n t s a n d s2p c o u n t s t h a t were f o u n d in t h e h e a v y position w a s e s t i m a t e d b y c u t t i n g o u t a n d w e i g h i n g t h e a r e a s f r o m a graph.

Biochim. Biophys. Aaa, 272 (I972) 312-32o

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practically no DNA has replicated as shown bv the lack of all-labeled DNA in tile heavy position. In contrast, hybrids formed between rRNA and DNA from each fraction of the density gradient, as represented by the 32p curve, are formed with DNA at the heavy position indicating that some of the rRNA genes have replicated. From the total amount of hybrids formed, it can be estimated that approx. I~ °i, of the total amount of rRNA genes are found in the heavy position. After I5o rain of refeeding, about 16 O//oof the total DNA has replicated as estimated from the relative amount of 3H label in the heavy position while the a2p curve indicates that about 45 ~/o of the rRNA genes have replicated. After 27 ° rain 72 ~o of the total DNA and 87 }o of the rRNA genes have replicated. We conclude from these data and data from later time points that replication of the rRNA genes has been initiated before replication of the bulk DNA. This is not a consequence of the partial synchronization of the DNA replication which is induced b y the starvation treatment '9 because it is known that the nucleolar DNA in Tetrahyrnena 26 (as in other organisins 27) is late replicating, i.e. the synthesis of nucleolar DNA normally takes place during the later part of and after the macronuclear S period. It is seen from Fig. 3 that at 270 rain after refeeding, the newly synthesized rRNA genes coincide in density with the newly synthesized bulk DNA .The rRNA genes do not coincide in density with bulk DNA when not substituted with BrdU (Fig. 2). This difference can be explained by the fact that bulk DNA has a lower GC content (i.e. higher AT content) than rRNA genes so that relatively more BrdU will be incorporated in the newly synthesized bulk DNA than in rRNA genes. The presence of BrdU in the growth medium does not induce a preferential synthesis of rRNA genes, neither does BrdU containing DNA hybridize selectively to rRNA. This was shown by an experiment where exponentially growing cells were allowed to take up BrdU for 2 h. When the nuclear DNA was isolated and used in hybridization experiments with increasing amounts of rRNA, this BrdU containing DNA reached a saturation value of 0.28 o~ /O • The saturation value obtained when unsubstituted DNA is used is o.32 o' / 0 (ref. 7)- When the newly svnthesized DNA containing BrdU (Fig. 3) was rebanded in an alkaline CsC1 density gradient, two separate DNA peaks were observed, one at the position of BrdU containing single stranded DNA and the other at the position of unsubstituted single stranded DNA. The lack of material of hybrid density indicates the semi-conservative nature of the replication that has taken place tl.

DISCUSSION The observation of preferential replication of rRNA genes after nutritional shift-up in Tetrahymena suggests t h a t replication of rRNA genes and replication of total nuclear DNA are under separate controls. This interpretation correlates with our previous observations that there is an approx. 30 % increase in the number of r R N A genes in exponentially growing Tetrahymena compared with starved or stationary phase cellsL Nucleolar r R N A genes are of particular interest for the regulation of rRNA synthesis. In addition to the mechanisms which control RNA synthesis at the transscriptional level 28, a specific replication of rRNA genes could provide an amplifying

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mechanism during periods of intensive rRNA synthesis. Such amplification of rRNA genes has been reported in the oocytes of certain amphibians, an echinoid worm and the surf clam .3. The classical theory of gene amplification .3'24'.9's° asserts that the rRNA genes are replicated m a n y times without concurrent replication of the rest of the genome in the same cell. The mechanism of the preferential replication of the rRNA g e n e s observed in this study m a y not differ in principle from the situation observed in oocytes. Replication of rRNA genes is initiated prior to replication of the rest of the nuclear DNA after nutritional shift-up. We emphasize, however, that our data do not suggest that rRNA genes are replicated m a n y times without replication of the rest of the genome. A specific rDNA amplification is not observed. Considering the small increase in the amount of rRNA genes during refeeding in Tetrahymena,it is not likely t h a t the increase in the amount of rRNA template can account for the subsequent rapid synthesis of rRNA. It seems more likely that some replication of the rRNA genes is a prerequisite for the switching on of the transcription of the rRNA. Some translation m a y also be important in regulating the synthesis of rRNA v. There still exists the possibility that the observations in Fig. 3 are a result of some kind of "false" initiation of the replication of the rRNA genes which is induced by the administration of fresh growth medium. In this case, the only conclusion t h a t can be made is that initiation of the replication of the bulk nuclear DNA and the nucleolar DNA are under separate controls.

ACKNOWLEDGEMENT

The authors are indebted to Dr Vagn Leick for helpful discussions and suggestions. This work was supported by grants from the National Research Council of Canada, the Defence Research Board of Canada and the Danish Research Council for Natural Sciences. D. M. is a predoctoral fellow of the National Research Council of Canada. REFERENCES I. L. Cameron and E. E. Guile, Jr, J. Cell Biol., 26 (1965) 845. I. L. Cameron, G. M. Padilla and O. L. Miller, Jr, f . Protozool., 13 (1966) 336. M. R. Levy and A. M. Elliott, J. Protozool., 15 (1968) 208. B. Satir and E. R. Dirksen, J. Cell Biol., 48 (1971) 143. J- R. Nilsson and V. Leick, Exp. Cell Res., 60 (197 o) 361. H. Busch and K. Smetana, The Nucleolus, Academic Press, New York and London, i97 o, p. 173-185. 7 J- Engberg and R. E. Pearlman, Eur. f . Biochem., in the press. 8 A. M. Elliott, L. E. Brownell and J. A. Gross, J. Protozool., I (1954) 193. 9 I. L. Cameron and J. R. Jeter, Jr, J. Protozool., 17 (197 o) 429. io Y. C. Lee and J. E. Byfield, Biochemistry, 9 (197 o) 3947. I I C. F. Brunk and P. C. Hanawalt, Science, 158 (1967) 663. 12 V. Leick, J. Engberg and J. Emmersen, Eur. J. Biochem., 13 (197o) 238. 13 V. Leick and P. Plesner, Biochim. Biophys. Acta, 169 (1968) 398. 14 A. P. Nygaard and B. D. I~all, Biochem. Biophys. Res. Commun., 12 (1963) 98. I5 F. Solymosy, G. Lazar and G. Bagi, Anal. Biochem., 38 (197 o) 4o. 16 D. Gillespie and S. Spiegelman, J. Mol. Biol., 12 (1965) 829. I 7 ~ . Birnstiel, J. Speirs, I. Purdom, K. Jones and U. E. Loening, Nature, 219 (1968) 45418 V. Leick, Eur. J. Biochem., 8 (1969) 221. I 2 3 4 5 6

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C. F. Brunk, P h . D . Thesis, Biophysics Laboratory RepoJ,t 207, Stanford I "nivcrsity, I9~)7, p. 5b C. L. Schildkraut, J. M a r m u r and P, J. Doty, jr. Mo/. Biol., 4 (1962) 43 °. R. A. Flavell and I. G. Jones, Biochem..]'., 116 (197 ° ) 81i. Y. S u y a m a , Biochemist[!~, 5 (1966) 2214. D. D. B r o w n and I. B. Dawid, Science, 16o (1968) 272. J. G. Gall, Proc. Natl. Acad. Sci. U.S., 60 (I968) 553I. Balazs and C. I,. Schildkraut, jr. ~Viol. Biol., 57 (1971) 153. R. Charret, Exp. Cell Res., 54 (1969) 353. I~. Busch and K. Smetana, The Nucleolus, Academic Press, N e w Y o r k and London, 197 o, p. 185. F. C. Neidhardt, in J. N. D a v i d s o n and VV. E. Cohn, Progr. Nucleic Acid Res. and Mol. Biol., Vol. 3, Academic Press, New Y o r k and London, 1964, p. 145. 29 E. Perkowska, H. C. MacGregor and 1Vf. L. 13irnstiel, Nature, 217 (1968) 649. 3 ° D. E v a n s and M. 1,. Birnstiel, Biochim. Biophys. Acta, 166 (1968) 274. 19 zo 2r 22 23 24 25 26 27 28

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