Hybridization properties of ribosomal RNA from rabbit tissues

I6 9

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96181

H Y B R I D I Z A T I O N P R O P E R T I E S OF RIBOSOMAL RNA FROM R A B B I T TISSUES A D E L E D I G I R O L A M O , E N Z O B U S I E L L O AND M A R I O D I G I R O L A M O

International Laboratory o/Genetics and Biophysics, Naples (Italy) (Received N o v e m b e r I ith, 1968)

SUMMARY

The capacity of 28-S and I8-S ribosomal RNA isolated from reticulocytes and liver to hybridize to DNA has been studied. The plateau values reached b y ribosomal RNA are the same, irrespective of whether ribosomal RNA from liver or from reticulocytes, or a mixture of both RNA's, are used. Moreover, ribosomal RNA from one tissue competes to form a hybrid to DNA with ribosomal RNA from the other tissue. These results have been obtained with both 28-S and I8-S ribosomal RNA and, on this basis, it has been concluded that the major base sequences of ribosomal RNA's in reticulocytes and liver are the same.

INTRODUCTION

Studies carried out in various laboratories on differences between ribosomes of different animal tissues have indicated that ribosomal proteins have identical electrophoretical behaviour 1-3, irrespective of their embryonic origin, degree of differentiation and functional specialization. The data concerning the RNA moiety of ribosomes are more contradictory. REICH et al. 4 found differences in base composition of ribosomal RNA prepared from different tissues, while HIRSH5 did not find any significant difference in the base composition of either 28-S or I8-S ribosomal RNA isolated from different tissues of the same animal. GOULD et al. ~ analyzed the products of partial digestion with T I ribonuclease of ribosomal RNA prepared from the reticulocytes and liver of rabbit and found no differences. In order to obtain information on the base sequence of ribosomal RNA of different tissues, we have analyzed the capacity of 28-S and I8-S ribosomal RNA isolated from reticulocytes and liver of rabbit to hybridize to DNA. These two tissues have been chosen since they are quite different both in embryological origin and in the proteins they are able to synthesize. The results presented in this paper show that the 28-S and I8-S ribosomal RNA's isolated from both tissues are indistinguishable in their capacity to hybridize to DNA. MATERIALS AND METHODS

Preparation o / 4 7 - S and 32-S sub-units

Rabbits weighing approx. 3 kg were used. Liver polysomes were obtained from rabbits starved 2 days before sacrifice. Reticulocyte polysomes were isolated from the Biochim. Biophys. dcta, 182 (1969) 169-174

17o

A. DI GIROLAMOet al.

peripheral blood of rabbits made anaemic with phenylhydrazine, according to the procedure of BORSOOK et al. ~. The polysomes were prepared according to WETTSTEIN et al. s. Monosomes were obtained b y treatment of polysomes with ribonuclease (o.05/~g/ml for 5 min at 37 °) in Tris-KC1 (0.05 M Tris, p H 7-5, 0.025 M KC1) and separated b y centrifuging the ribonuclease-treated particles at 24 ooo rev./min for 2 h on a lO-4O °/o (w/w) linear sucrose gradient. Fractions corresponding to the monosome peak were pooled and centrifuged overnight at 40 ooo rev./min. For the preparation of 47-S and 32-S sub-units, 3.5-5 mg of monosomes, obtained as described above, were resuspended in I mM Tris, pH 7.5, 5 ° mM KC1 buffer and treated with E D T A (2.5 #moles/mg ribosome). The ribosomal sub-units 9 were separated on a 5-20 °/o; (w/w) sucrose gradient in the same buffer for 8 h at 24 ooo rev./min. The fractions corresponding to the peaks of 47-S and 32-S sub-units were pooled and centrifuged overnight at 4 ° ooo rev./min. It should be noted that the 47-S sub-unit peak in preparative sucrose gradient was consistently asymmetrical. It has been shown 3 that this peak can be broken down by a Monte Carlo method 1° into three Gaussian components with sedimentation constants of 47 S, 53 S and 60 S. In order to obtain uncontaminated 47-S particles, only the fractions corresponding to the tip of the peak were pooled and used in this study.

Preparation and purification o / R N A RNA was extracted from 47-S and 32-S sub-units by cold phenol extraction and precipitated b y addition of NaC1 and ethanol, as previously described n. The 28-S and I8-S RNA preparations were dissolved in o.I M NaC1, 0.05 M phosphate (pH 6.8) buffer and loaded at 50/~g/ml or less on methylated albumin Kieselguhr columns, prepared according to MANDELL AND HERSHEY1~. The elution was accomplished with linear NaC1 gradients from o.I M to 1. 5 M NaC1 in 0.05 M phosphate buffer. The total eluting volume was 200 ml and 5-ml fractions were collected. The resulting purified 28-S and I8-S RNA fractions were pooled, dialyzed overnight against o.I M NaC1, precipitated b y 2 vol. ethanol and washed once with 80 °/o ethanol. Labeling o] ribosomal R N A 5 mC (6.3 rag) of EaH]dimethyl sulfate were added to o.5 mg of RNA in i ml of o.I M phosphate buffer (pH 7.5) and the mixture was incubated for 2 h at room temperature. The excess of dimethyl sulfate was removed by repeated extraction with benzene. After addition of NaC1 to a final concentration of o.I M, the RNA was precipitated with 2 vol. of ethanol. The RNA, dissolved and precipitated a second time with ethanol, was washed twice with 80 °/o ethanol and then dialyzed against o.15 M NaCl-o.oI 5 M trisodium citrate. Preparation and denaturation o / D N A The DNA was extracted from rabbit liver nuclei according to the MARMUR13 procedure, as modified by RITOSSA AND SPIEGELMAN14. Alkaline denaturation of DNA was carried out in 1.5 mM NaCl-o.I5 mM trisodium citrate at a concentration of lOO-15o/,g/ml following exactly the method described by GILLESPIE AND SPIEGELMAN 15.

Biochim. Biophys. Acta, 182 (1969) 169-174

RIBOSOMAL

RNA OR RABBIT TISSUES

171

Hybridization with immobilized DNA The R N A - D N A hybridization procedure was essentially that described by GILLESPIE AND SPIEGELMAN 15. To detect the degree of "noise", a blank filter, containing in some experiments immobilized Escherichia coli DNA, was included in each incubation vial and was subiected to all steps of the procedure. The amount of radioactivity found on this filter provided the measure of the noise level. This was rather high, around o.i % of radioactivity of RNA input.

RESULTS

In order to prepare pure 28-S and I8-S ribosomal RNA, the RNA was extracted from 47-S and 32-S sub-units derived from monosomes obtained by treating polysomes with ribonuclease. Under these conditions, only m R N A was degraded and any m R N A still remaining could be easily removed during separation of the sub-units on the sucrose gradient. The ribosomal RNA extracted from the sub-units was further purified on a methylated albumin column. Since we were not able to obtain ribosomal RNA with a specific activity higher than 300 counts/min per Fg by injecting intraperitoneally up to 5 mC/kg of E32P~ortophosphate in the rabbit, we labeled the ribosomal RNA in vitro with [aH]dimethyl sulfate 1~.

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Fig. i. H y b r i d i z a t i o n of 28-S r i b o s o m a l R N A w i t h D N A . A n n e a l i n g process was p e r f o r m e d a t 60 ° for 12 b. B e t w e e n 55 a n d 65/~g of D N A were loaded onto m e m b r a n e filters. T h e specific a c t i v i t y of 28-S r i b o s o m a l 1RNA f r o m liver was 55oo c o u n t s / m i n p e r / , g a n d f r o m r e t i c u l o c y t e s was 6ooo c o u n t s / m i n per/~g. A - & , 28-S labeled r i b o s o m a l R N A f r o m liver; × - x , 28-S labeled r i b o s o m a l R N A f r o m reticulocytes; O - O , m i x t u r e of 28-S labeled r i b o s o m a l R N A f r o m liver a n d r e t i c u l o c y t e s (ratio I : I). Fig. 2. H y b r i d i z a t i o n of I8-S r i b o s o m a l R N A w i t h D N A . A n n e a l i n g process was p e r f o r m e d as described u n d e r Fig. I. T h e specific a c t i v i t y of I8-S r i b o s o m a l R N A f r o m liver a n d r e t i e u l o c y t e s w a s 61oo c o u n t s / m i n p e r /~g a n d 65oo c o u n t s / m i n p e r #g, respectively. & - A , I8-S r i b o s o m a l 1RNA from liver; x - x , I8-S r i b o s o m a l R N A f r o m reticulocytes; O - O , m i x t u r e of I8-S labeled r i b o s o m a l R N A f r o m liver a n d r e t i c u l o c y t e s (ratio i : i ) .

28-S ribosomal RNA isolated from reticulocytes and liver was incubated at various input levels with a constant amount of DNA immobilized on a nitrocellulose filter. The results reported in Fig. I show that the plateau value reached is the same (approx. 0.06 %) for both tissues. Moreover, if 28-S ribosomal RNA from reticulocytes and liver was incubated simultaneously with DNA, the amount of ribosomal RNA which hybridizes with DNA does not change. Biochim. Biophys. Acta, 182 (1969) 169-174

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TABLE I PERCENT

RNA

TO

D N A IN

HYBRIDS BETWEEN

DNA

AND RIBOSOMAL

Hybrid

RNA

RNA /DNA

(%) 28-S 28-S I8-S I8-S 28-S I8-S 28-S

RNA RNA RNA RNA RNA RNA RNA

from from from from from from from

liver reticulocytes liver reticulocytes l i v e r + 2 8 - S R N A from reticulocytes l i v e r + I8-S R N A from reticulocytes l i v e r + I8-S R N A from liver

0.06 0.06 0.03 0.03 0.06 0.03 0.o8

The s a t u r a t i o n curves for I8-S r i b o s o m a l R N A ' s from b o t h tissues were also similar a n d no change in t h e p l a t e a u value was found when the R N A ' s from t h e two tissues were h y b r i d i z e d together. The results of the various e x p e r i m e n t s are

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3'0

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20

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"~ 200 100

Fig. 3. Competition of ribosomal R N A from different tissues to form a h y b r i d with DNA. Annealing process w a s performed as described u n d e r Fig. i. The specific activity of labeled ribosomal R N A was 5500 c o u n t s / m i n per /~g for 28-S R N A f r o m liver, 60o0 c o u n t s / m i n per /*g for 28-S R N A f r o m reticulocytes, 61oo c o u n t s / m i n p e r / ~ g for I8-S from liver, 6500 c o u n t s / m i n p e r / z g for I8-S from reticulocytes. (a) Competition w i t h I8-S labeled R N A from liver. 60/zg of D N A were loaded on m e m b r a n e filters. A - A, H y b r i d i z a t i o n w i t h I8-S labeled R N A from liver; × - × , hybridization with m i x t u r e I8-S labeled R N A from liver and unlabeled I8-S R N A f r o m liver (ratio i : i ) ; A - A , hybridization with m i x t u r e I8-S labeled R N A from liver and unlabeled I8-S R N A from reticulocytes (ratio i :i). (b) Competition w i t h I8-S labeled R N A from reticulocytes. 55/*g of D N A were loaded on m e m b r a n e filters. A - A , Hybridization w i t h I8-S labeled R N A from reticulocytes; × - × , hybridization w i t h m i x t u r e I8-S labeled R N A from reticulocytes and unlabeled I8-S from reticulocytes (ratio I : I ) ; A - A , hybridization with m i x t u r e I8-S labeled R N A from reticulocytes and unlabeled I8-S R N A from liver (ratio i : i ) . (c) Competition with 28-S labeled R N A from liver. 5 ° /2g of D N A were loaded o n t o m e m b r a n e filters. A - A , Hybridization w i t h 28-S labeled R N A from liver; × - × , hybridization w i t h m i x t u r e 28-S labeled R N A f r o m liver and unlabeled 28-S R N A from liver (ratio i : i ) ; A - A , hybridization with m i x t u r e 28-S labeled R N A from liver and unlabeled 28-S R N A f r o m reticulocytes (ratio I : I ) . (d) Competition with 28-S labeled R N A from reticulocytes. 5 ° # g of D N A were loaded onto m e m b r a n e filters. A - A , Hybridization with 28-S labeled R N A from reticulocytes; × - X , hybridization w i t h m i x t u r e 28-S labeled R N A from reticulocytes and unlabeled 28-S R N A from reticulocytes (ratio i : i) ; A - £x, hybridization with m i x t u r e 28-S labeled R N A from reticulocytes and unlabeled 28-S R N A from liver (ratio i :I).

Biochim. Biophys. Acta, I82 (1969) I69-I74

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RNA

OR RABBIT TISSUES

173

summarized in Table I. I t can be seen that the plateau values for 28-S and I8-S ribosomal RNA are 0.06 °/o and 0.03 °/o, respectively, in both liver and reticulocytes, and remain unchanged when ribosomal RNA from both tissues are hybridized together. However, when a mixture of 28 S and 18 S is hybridized, the plateau value found corresponds roughly to the sum of the saturation values of 28-S and I8-S ribosomal RNA. When labeled ribosomal RNA from one tissue is mixed in I : I ratio with unlabeled ribosomal RNA from the other tissue, the amount of labeled RNA hybridized to DNA is reduced to one half. This is valid for both 28-S and I8-S ribosomal RNA, as shown in Fig. 3.

DISCUSSION

The problem we tried to answer was whether ribosomal RNA from different tissues of the same animal is different in base sequence. For this purpose we analyzed the capacity of 28-S and I8-S ribosomal RNA isolated from reticulocytes and liver to hybridize to DNA. The purity of RNA preparations is essential for this type of study. Care was taken to eliminate any m R N A contamination or cross-contaminanation between 28 S and 18 S. In a previous paper 17, we demonstrated that if ribosomal RNA is isolated from monosomes obtained b y ribonuclease treatment of polysomes, the small amount of m R N A still presents sediments in a much lighter region and can be easily separated. In another paper 3, we have shown that it is possible to isolate 47-S ribosomal subunits contaminated with less than 1. 5 ~o of the 32-S component and the smaller ribosomal sub-unit uncontaminated by the larger one. On the basis of these previous results, we have isolated 28-S and I8-S ribosomal RNA from uncontaminated subunits derived from monosomes obtained b y treatment of polysomes with ribonuclease. Moreover,we have further purified the RNA by means of a methylated albumin column. We feel confident that the ribosomal RNA isolated by this procedure does not contain any contamination. Since the labeled ribosomal RNA, which we could obtain by labeling i n vivo, had a specific activity of less than 300 counts/rain per #g, we labeled the RNA in vitro with E3H]dimethyl sulfate. I t has been shown by SMITH et al. 18 that RNA labeled in this way has hybridization properties identical to RNA labeled in vivo. We thus obtained ribosomal RNA with specific activity about 60o0 counts/nfin per/~g, which is still low (and in fact the experimental variability was rather high) but sufficient to give consistent results. The hybridization experiments reported in this paper show that ribosomal RNA isolated from liver and reticulocytes gives the same plateau value in each case, does not present additivity and competes in the presence of each other preparation. All these results lead to the conclusion that the major sequences of both 28-S and I8-S ribosomal RNA's in reticulocytes and liver are the same, but the possibility that small differences in base sequences do exist cannot be ruled out since the hybridization technique is not sufficiently sensitive to evidence them. Since reticulocytes and liver are very different tissues, both in embryological origin and in the proteins they are able to synthesize, we feel that our conclusions JBiochim. Biophys. Acta, 182 (1969) 169-174

174

A. DI GIROLAMO ¢~

al.

can be generalized for all other tissues. The data presented here are also in basic agreeement with results that have appeared while this work was in progress, which show (a) 28-S ribosomal RNA isolated from sea urchin embryos at various developmental stages compete with the same piece of DNA to form a hybridlS; (b) ribosoma] RNA's from various human tissues have the same base composition, give rise by mild TI ribonuclease digestion to polynucleotides with the same sedimentation constant, release by pancreatic ribonuclease digestion the same amount and type of mono-, di- and trinucleotides, and compete in hybridization experiments with the same piece of DNA (F. AMALDI AND G. ATTARDI, personal communication). Our control experiments, showing additivity in the plateau values of 28 S and 18 S, also confirm the existence of different base sequences between 28 S and 18 S. Furthermore the high plateau values obtained for ribosomal RNA from rabbit tissues also suggest the existence of a redundancy of genes for ribosomal RNA in this organism. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18

GIUDICH AND E. MUTOLO, Biochim. Biophys. Acta, 138 (1967) 214. B. L o w AND I. G. WOOL, Science, 155 (1967) 33 o. DI GIROLAMO AND P. CAMI~IARAN0, Biochim. Biophys. Acta, 168 (1968) 181. :REICH, G. ACS, B. MECH AND E. L. TATUM, in H. J. VOGEL, V. BRYSON AND J. O. LAMPDEN, In[ormational Macromolecules, Academic Press, New York, 1963, p. 317 . C. A. HIRSH, Biochim. Biophys. Acta, 123 (I966) 246. A. GOULD, S. BONANOU AND K. KANAGALINGHAM,J. Mol. Biol., 22 (i966) 397. H. N. BORSOOK, E. FISCHER AND G. KEIGHLEY, J. Biol. Chem., 229 (I957) lO59. F. O. WHTTSTEIN,T. STAEHELIN AND H. NULL, Nature, 197 (1963) 43 o. Y- TASHIRO AND P. SIEKEVITZ, .]. Mol. Biol., I I (1965) 149. G. CORTELLESSA AND G. FARCHI, Rept. Ist. Super. Sanit. Rome, 65 (1965) IO. H. H. HIATT, J. Mol. Biol., 5 (1962) 217. J. D. MANDELL AND A. D. HERSHEY, Anal. Bioehem., i (196o) 66. J. MARMUR, J. Mol. Biol., 3 (1961) 208. F. M. RITOSSA AND S. SPIEGELMAN,Proc. Natl. Acad. Sci. U.S., 53 (1965) 737. D. GILLHSPIE AND S. SPIEGELMAN,]. Mol. Biol., 12 (1965) 829. K. D. SMITH, J. L. ARMSTRONG AND B. J. McCARTHY', Biochim. Biophys. Aeta, 142 (1967) 323 ` A. DI GIROLAMO, M. DI GIROLAMO, S. GAETANI AND M. A. SPADONI, European J. Biochem., I (1967) 164 . G. GIUDICH AND E. MUTOLO,Biochim. Biophys..4cta, 149 (1967) 291. G. R. M. E.

Biochim. Biophys. Acta, 182 (1969) I69-I74