Studies on ribosomal RNA from tetrahymena by zone velocity sedimentation in sucrose gradients and base ratio analysis

320

BIOCHIMICA ET BIOPHY,qlCA ACTA

BBA 96258

S T U D I E S ON RIBOSOMAL RNA FROM T E T R A H Y M E N A BY ZONE VELOCITY S E D I M E N T A T I O N IN SUCROSE G R A D I E N T S AND BASE RATIO ANALYSIS A J I T KUMAR*

Department o] Biology, University o/ Chicago, Whitman Laboratory, 9z5 E. 57th Slreel Chicago, Ill. 6o637 (U.S.A.) (Received March I2th, 1969)

SUMMARY

A method for the isolation of ribosomal RNA (rRNA) from Tetrahymena is described. Purified rRNA, when analyzed on sucrose density gradients zonal centrifugation, showed two main peaks sedimenting at about 25.4 S and 17 S. Base ratio analysis showed low (36 %) G + C mole %. On the basis of the sedimentation rate, as well as the low G + C content, it is suggested that Tetrahymena rRNA is unlike the known forms of either prokaryotes or higher eukaryotes and possibly represents an intermediate form.

INTRODUCTION

The central role of ribosomes in bringing together components of the protein biosynthetic apparatus in cells has led to an increasing interest in these particles. A number of recent reports on both lower and higher forms have discussed ribosomal structure and formation (see refs. I and 2 for review), but m a n y details, such as the specific role of ribosomal RNA (rRNA) remain unanswered. A comparative study of rRNA is relevant both in the analysis of ribosomal structure and of its variability in evolution. Distinctions among ribosomes have recently been proposed on the basis of the size of rRNA molecules, but since the number of carefully studied organisms 3-6 is limited, broad evolutionary trends are not yet clearly evident. The present report describes sucrose density gradient analysis of RNA in the protozoan Tetrahymena pyri/ormis. The larger of the two rRNA's was found to sediment at 25.4 S and the smaller one at 17 S. The larger T e t r a h y m e n a rRNA component, at least on the basis of its sedimentation rate, seems closer to phytoflagellates (25 S) 7, fungi (24 S) 5 and higher plants (25 S) ~'° than to higher animals (28 S) 8. The smaller rRNA (17 S), on the other hand, seems to differ less from its counterpart in prokaryotes ( I 6 S) 5. The base ratio analysis of purified T e t r a h y m e n a rRNA showed a low G + C content. MATERIALS AND METHODS

Culture conditions Cultures of T. pyri/ormis (mating Type i, variety I) were grown in 2 % proteose peptone (Difco) supplemented with 0.2 % glucose, 0.i % yeast extract and 0.003 % " P r e s e n t address: D e p a r t m e n t of Biochemistry, Albert E i n s t e i n College of Medicine, t3oo Morris P a r k Avenue, Bronx, N. Y. 10461, U.S.A.

Biochim. Biophys. dcla, i86 (t969) 326-33 t

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sequestrene ( F e - E D T A , Geigy) (F. M. CHILD, personal communication). Axenic cultures were maintained at 25 ° with constant aeration. The average generation time under these conditions was about 7 h. Ribosome isolation Cells from log-phase cultures were collected b y centrifugation at 125o × g • rain and were washed in 15 mM Tris-HC1 buffer ( p H 7.5) at room temperature; 5 mM KC1; 1.5 mM MgC12 (Tris-HC1-KC1-MgC12 buffer), supplemented with 0.25 M sucrose and o.oi 5o spermidine • HC1. W a s h e d cells were suspended in IO vol. of the above buffer and were lysed b y passing through a narrow orifice under pressure in a Logeman h a n d mill (Logeman Co., Chicago). The duration of the t r e a t m e n t was about 5 sec at 2-4 °. After three repetitions, no whole cells were detectable upon light microscope examination, whereas most of the nuclei remained intact. The large particulate fractions were removed from the cell lysate b y centrifugation at 16 ooo × g for 20 rain at 2 °. Ribosomes were pelleted from the supernatant at lO5 ooo × g for 9 ° min at 2 °. R N A extraction Solutions: A, o.15 M sodium acetate, o.i M NaC1, i °/o sodium dodecyl sulfate; B, 6 °//o (w/v) sodium 4-aminosalicylate; C, Phenol-cresol mixture: 500 g phenoldetached crystals, 7 ° m l m-cresol (Eastman Chemicals), 55 ml water, 0.5 g 8-hydroxyquinoline. The procedure is based on the second phenol-cresol m e t h o d of KIRBY9. Cells from log-phase culture were collected and washed in Tris-HC1-KC1-MgC12 buffer supplemented with 0.25 M sucrose and o.oi % spermidine at 2000 × g • rain (or 200 × g for IO rain) at 2-4 °. The washed cell pellet was immediately frozen in solid CO 2acetone b a t h until used for R N A extraction. The frozen cell pellet was homogenized in 5 times its volume of Sol. A. The homogenate was supplemented with an equal volume of Sol. B, and 2 vol. of Sol. C and extracted for 25 rain. The phenol phase was separated b y centrifugation at IOOO × g for IO rain. The aqueous phase was carefully removed and NaC1 was added to a final concentration of 2 %; the mixture was reextracted with 0.5 vol. of Sol. C for 15 rain. R N A was precipitated from the final aqueous phase with 2 vol. of 80 °/o ethanol, containing i °/o NaC1 for at least 2 h in the cold (--20°). The R N A precipitate was collected b y centrifugation and was washed with cold 3 M sodium acetate (pH 6.5) to remove DNA, glycogen and low molecular weight R N A 9. The r R N A pellet was then washed once with 95 % ethanol and once with IOO o~) ethanol. Tile concentration of the R N A samples dissolved in Tris-HC1-KC1MgC12 buffer was determined b y its absorbance at 260 m/~ (20 A2n0 m# units = I rag). H e L a cells were grown in suspension to a concentration of 4" lO5 cells per ml in minimal essential m e d i u m 1° with IO °/o calf serum. R N A was labeled b y adding o.I #C/ml (3H]uridine (specific activity 20 C/mole) to the culture for 48 h. Cells were collected b y centrifugation at 800 × g for 2 rain and washed once in H a n k s saline solution n. R N A from whole cells was extracted b y the m e t h o d described for Tetrahymena. Linear sucrose density gradients (15-3o % w/v) were made in Tris-HC1-KC1MgC12 buffer. The details of the centrifugation conditions are described in the explanation of figures. Gradients were eluted through a Gilford continuously recording Biochim. Biophys. Acta, 186 (1969) 326-33t

328

A. KUMAI¢

spectrophotometer and were collected in o.7-ml fractions. Trichloroacetic acid was added to the radioactive fractions to a final concentration of Io ~}~o.The precipitate (without added carrier) was collected on Whatman (GF/A) glass filters, was washed several times with 5 % trichloroacetic acid followed by 7o% ethanol. Dried filters were counted in I : 2o dilution of Liquifluor (5° g 2.5-diphenyloxazole; o.625 g 1.4-bis-(5-phenyloxazolyl-2)benzene in toluene, in a Packard liquid scintillation spectrometer. Base ratio analysis were made by the method of KATZ and COMB~2, except that CMP was eluted from Dowex-I (formate) columns with o.I M formic acid instead of o.o5 M formic acid as mentioned in their paper (K. G. NAIR, personal communication).

RESULTS

The sedimentation rate of purified Tetrahymena RNA was estimated by direct comparison with Escherchia coli and HeLa cell RNA in sucrose density gradients. As a test of validity for such comparisons, the distance moved by the RNA molecules through sucrose gradients in time should bear a linear relationship 1~. Results from several independent experiments (data not illustrated) showed that the conditions used to compare Tetrahymena RNA with known forms of RNA as markers, satisfied such requirement. On co-sedimentation of unlabeled Tetrahymena RNA with 3Hlabeled E. coli rRNA, the smaller E. coli rRNA sedimented close to the slower sedi-

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Fig. I. Compxrison of s e d i m e n t a t i o n r a t e s of T e t r a h y m e n a r R N A w i t h E. coli r R N A . Unlabeled T e t r a K y m e n a R N A was layered on a 3o-ml sucrose gradient (15-3o °/o w / v in Tris-HC1-KC1-MgC1 z buffer) along w i t h o. 5 % [3H]uracil-labeled E. cell r R N A , and centrifuged in the 25.1 rotor on a Spinc~ Model L ultracentrifuge at 25 ooo r e v . / m i n for 13. 5 h at 6 °. SH c o u n t s in broken line.

menting Tetrahymena rRNA (Fig. I). On the basis of the published values for sedimentation rates of E. coli rRNA (23 S and 16.7 S) 14, the relative sedimentation rates for Tetrahymena rRNA were estimated to be 25.4 S and 17 S. On a similar comparison of Tetrahymena RNA with HeLa cell RNA (28 S and 16 S) 15, as illustrated in Fig. 2, Biochim. Biophys. dcta, 186 (I969) 326-331

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Fig. 2. C o m p a r i s o n of s e d i m e n t a t i o n rate of T e t r a h y m e n a r R N A w i t h H e L a r R N A . Unlabeled T e t r a h y m e n a R N A w a s layered on a 3o-ml sucrose gradient ( I 5 - 3 o % in 5 mM Tris-HC1 (pH 7-4); o.i M NaCI; o.i mM E D T A and 0. 5 % s o d i u m dodecyl sulfate) along w i t h 6 % [aH]uridinelabeled H e L a cell R N A and centrifuged at 2 5 ooo r e v . / m i n for 14.2 5 h at 17 °.

the smaller T e t r a h y m e n a rRNA sedimented close to the I6-S H e L a rRNA, whereas the larger Tetrahymena rRNA sedimented distinctly slower than the 28-S H e L a cell rRNA. As reported earlier 9, the sedimentation rates of the two rRNA's (Figs. I and 2) are unaffected by 3 M sodium acetate extraction of the crude RNA preparation and the two rRNA peaks are entirely sensitive to mild ribonuclease treatment. The base ratio analysis of unpurified total cell RNA was compared with rRNA separated on sucrose gradients (Table I). The observed mole % G + C for the whole cell RNA (43-5 %) was in agreement with an earlier report obtained from unpurified ribosomal extract. However, a lower value of G + C mole % (35.8 %) was observed TABLE I BASE RATIO ANALYSIS FOR TETRAHYMNNA AND E. coli r R N A Whole cell R N A s a m p l e s f r o m which r R N A w a s n o t purified w i t h cold 3 M s o d i u m acetate extraction (see text). Whole cell rlRNA precipitate w a s w a s h e d w i t h cold 3 M s o d i u m acetate to r e m o v e DNA, glycogen and low molecular w e i g h t R N A (see text). The 25.4-S and I7-S R N A samples were s e p a r a t e d on sucrose gradients. Pooled fractions were dialyzed a g a i n s t 5o0 vol. of Tris-HC1-KC1MgCI~ buffer, rapidly e v a p o r a t e d to reduce the v o l u m e to 0.5 ml and hydrolyzed for 18 h a t 37 ° in o. 3 M K O H . E. coli r R N A : r R N A f r o m K I 2 cells w a s p r e p a r e d and the base ratio analysis done b y the m e t h o d s used for T e t r a h y m e n a r R N A . Results are expressed as mole %.

Nucleotides Whole cell R N A

UMP GMP CMP AMP

rRNA

25.4-S r R N A

I7-S r R N A

Prep. A Prep. B

Prep. A Prep. B

Prep. A Prep. B

Prep. A Prep. B

31.7

32.1 12.3 22. 4 33 .0

33.9 16.3 15.2 34.4

32.7 19.8 16.2 31-2

23.2 19.6 25.3

28.4 22.3 21. 9 27.3

29.3 15.7 21. 3 33 .6

3o-3 17.3 17.1 35 .1

3o.8 21-3 15.3 32.5

E. coli rRNA

2o.8 33.3 21.6 24 .2

Biochim. Biophys. Acta, 186 (1969) 326-331

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from purified rRNA sample. An earlier report on the base composition of RNA fr~m the amicronucleate strain T. p y r i / o r m i s GL also showed a low G + C content (39 '!,,)The higher value of CMP for rRNA (Table I) as compared to that of the 25.4-S RNA probably represents an error in determining the effluent volume.

DISCUSSION

The results described here differ somewhat from two other recent studies on T e t r a h y m e n a ribosomes. SUYAMA18 reported that the amount of the larger rRNA was very low, only about Io °/o of the smaller component. WELLER et al. 19 found a prominent peak at 26 S, but the smaller component was only about 2o °/o of the larger and was considered to be 14 S. The authors suggest the large amount of 4-S material obtained was probably due to degradation during preparation. This might also produce the lower s value found for the smaller rRNA component. A possible reason for the loss m a y have been the action of ribonucleases and other enzymes which lead to the breakage of phosphodiester bonds. In an attempt to minimize such degradations in the present study, emphasis was placed on near neutral p H working conditions and rapid RNA extraction at low temperatures. Since the known form(s) (J. S. ROT~, personal communication (1966))of ribonuclease in Tetrahymena have an acid pH optimum 2°,21, the purified RNA samples were run in sucrose gradients made in Tris-HCI-KCI-MgC12 buffer (pH 7-5)- The effects of ions and temperature on the sedimentation properties of high polymer RNA's is quite complex and probably depends upon the nature of the RNA studied (see refs. 3, 22-25 for discussion). In this study, T e t r a h y m e n a RNA was directly compared with the known forms of rRNA's under different conditions. In absence of the MgC12, and in o.I M NaC1 at I7 °, Tetrahymena RNA showed marked increase of the faster sedimenting material above 25.4 S, as well as a small increase in the light material towards the top of the gradient (Fig. 2). At lower temperature (6°), and in 1.5 mM MgC1 without any added NaCI (Figs. i and 3), however, the rRNA peaks were consistently better resolved. There is increasing evidence in the literature to support the hypothesis that prokaryotes and certain eukaryote cell organelles, i.e., chloroplasts and mitochondria, contain rRNA of the 23-S and I6-S type, while the eukaryotes contain 25-S and I7-S rRNA (plants) or 29-S and I8-S rRNA (mammals) 3,~3,4,25,~. The reports on the nature of rRNA from lower eukaryotes demonstrate a wide range of sedimentation values. For example, the phytoflagellates, Englena (24-S and 2o-S rRNA 4) and Clamydomonas (24-S and I6-S r R N A 7) show a large difference in the size of the smaller rRNA. In a recent report, the rRNA of Paramecium (a ciliate related to Tetrahymena) was shown to be 25 S and 18 S (ref. 26). In the present study, the rRNA from Tetrahymena was directly compared with E. coli rRNA and H e L a cell RNA under various conditions, and the estimated sedimentation values were 25.4 S and 17 S. The available evidence on the rRNA from lower eukaryotes is not sufficient to permit one to suggest a possible evolutionary trend of rRNA, if any 2~. However, it is interesting to note that the sedimentation properties of the large rRNA from lower eukaryotes is closer to that of plants (25 S) than higher animals (28 S). The smaller RNA on the other hand, seems to show more variability (17 S in Tetrahymena to 2o S in Euglena). The significance of such a variability is not clear. Biochim. Biophys. Acla, 186 (1969) 326-33I

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ACKNOWLEDGMENTS

I am thankful to Dr. H. SWIFT for his valuable suggestions and support throughout the course of this work and to Dr. PETER GEIDUSCHECK for donating labeled E. coli rRNA. I wish to thank Drs. M. GOROVSKYand J. GREENBERG for their valuable discussion of the manuscript. Supported by U.S. Public Health Service Grants HD-I612 and Training Grant HD-I74. REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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Biochim. Biophys. Acta, 186 (I969) 326-331