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The purification and properties of ribosomes from the thymus nucleus
BIOCHIMICA ET BIOPHYSICA ACTA
840
THE PURIFICATION AND PROPERTIES OF RIBOSOMES FROM THE THYMUS NUCLEUS A. O. POGO*, B. G. T. POGO, V. C. LITTAU, V. G. ALLFREY AND A. E. MIRSKY The Rocke[eller Institute, New York, N . Y . (U.S.A.) AND
M. G. HAMILTON The Sloan-Kettering Institute New York, N . Y , (U.S.A.)
(Received November 27th, 1951)
SUMMARY Methods are described for the isolation and purification of ribonucleoprotein particles (ribosomes) from isolated calf-thymus nuclei. About half of the total ribonucleic acid of the nucleus can be recovered in the purified ribosome fraction. Nuclear ribosomes can be purified by centrifugation in a sucrose density-gradient. The ribosome peak contains about 36 % RNA. Treatment of the peak material with deoxycholate removes much of the protein. The resulting ribosomes have a sedimentation coefficient of 78 S and about 6o % of their mass is RNA. The average base composition of this ribosomal RNA is given and compared with that of other nuclear RNA fractions. The changes in state of the nuclear 78-S ribosome with changing pH, ionic strength, and Mg2+ concentration are described. Electron microscopy of isolated ribosomes after shadow-casting with chromium indicates that the 78-S ribosomes are about 200 A in width.
INTRODUCTION Previous studies of the mechanism of protein synthesis in isolated thymus nuclei have shown that amino acid incorporation into nuclear proteins involves the participation of ATP and amino acid "activating" enzymes1, 2, "transfer"-ribonucleic acidsZ, 3, and ribonucleoprotein particles, or ribosomes 2,*. The ribosome fractions prepared from isolated cell nuclei by differential centrifugation have been found to comprise a heterogeneous mixture of particles of differing composition, sedimentation properties, and metabolic activity 4. It is the aim of the present paper to (a) describe the extraction conditions leading to optimal yields and preservation of structure of thymus-nuclear ribosomes, (b) to present methods for the purification of nuclear ribosomes by centrifugation in a Abbreviations: RNP, ribonucleoprotein particles; TCA, trichloroacetic acid; DOC, deoxycholate. * Present address: Instituto de Biologia Cellular, Universidad Nacional de Cordoba, Casilla Correo 362, Cordoba (Argentina). Biochim. Biophys. Acta, 55 (1962) 849-864
850
A.O. POGO, et al.
sucrose density gradient, (c) to summarize studies of the effects of detergents (Lubrol W and sodium deoxyholate) on ribosome structure, and (d) to present the composition and sedimentation properties of highly purified nuclear ribosome fractions, as well as their appearance under the electron microscope. In a subsequent paper s, the incorporation of [14Clamino acids into different components of these purified ribosomes will be described, together with kinetic evidence that protein synthesized in nuclear ribosomes acts as precursor to the proteins in the soluble phase of the nucleus.
Extraction o/ nuclear ribosomes The presence of granules lOO-300 A in diameter within the thymocyte nucleus is evident in electron micrographs of thymus tissue sections 4. The granularity of the nucleus is evident even at relatively low magnifications (Fig. I). Particles of similar magnitude have been observed previously in nucleoli 6-s, on the lateral loops of lampbrush chromosomes 9, in association with nuclear membranes1°, 11, on the chromosomal rings of BALBIANITM, and distributed throughout the nuclear sap4, ~S. When isolated thymus nuclei are extracted with neutral buffer or saline solutions the particles are removed together with other proteins of the soluble phase of the nucleus4,14,~5; the particles can then be recovered from the extract b y centrifugation at high speeds. They have been found to contain a large proportion of ribonucleic acid4,15. The evidence that ribonucleoprotein particles so prepared are indeed derived from the nucleus is based not only on electron microscopy before and after extraction 4, but also on chemical and morphological evidence for the purity of the nuclear preparations employed in their isolation, and on functional tests which indicate that RNP isolated after labeling experiments with 14C amino acids must have been present inside the nucleus at the time of ~ac uptake. For example, it has been shown that thymus-nuclear ribosomes require a sodium environment for their activity in situa, ~6, whereas free cytoplasmic ribosomes are strongly dependent on potassium ions ~. Therefore, amino acid incorporation observed in a potassium-poor, sodium-rich medium is not likely to be due to cytoplasmic ribosomes adsorbed on the outer membrane of the isolated nucleus. Moreover, protein synthesis by the ribosomes of the nucleus ceases when the nuclei are treated with deoxyribonuclease, but not when ribonuclease is added to the medium4,14. Since intranuclear ribosomes are not susceptible to attack by ribonuclease as long as they remain inside the nucleus, while extranuclear or cytoplasmic ribosomes are readily disrupted by pancreatic RNAase4, is this enzyme effect allows an unequivocal test for the presence and activity of ribosomes within the cell nucleus. The extraction of nuclear ribosomes was originally carried out in 0.I M phosphate buffer ~4 or Tris buffers at p H 7.1 (see ref. 4). Subsequent work indicates that the pH, ionic strength, and Mg 2+ ion concentration of the extracting medium all affect the state and recovery of nuclear RNP particles. These variables were then investigated systematically with a view to selecting conditions which allow a maximum yield and optimal preservation of structure of ribosomes in the nuclear extract.
Isolation o/ nuclei Nuclei were isolated from fresh calf thymus tissue following the methods deBiochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
85I
Fig. I. E l e c t r o n m i c r o g r a p h of t h y m u s cells in t i s s u e section. T h e sections were fixed for i h in b u f f e r e d o s m i u m tetroxide, e m b e d d e d in m e t h a c r y l a t e a n d s t a i n e d w i t h u r a n y l acetate. N o t e t h e s m a l l e l e c t r o n - d e n s e g r a n u l e s w i t h i n t h e nuclei; t h e s e d i s a p p e a r w h e n t h e n u c l e a r r i b o s o m e s a r e e x t r a c t e d in Tris b u f f e r s as described in t h e t e x t . T h i s electron m i c r o g r a p h w a s t a k e n b y B. S. McEWEN of t h e Rockefeller I n s t i t u t e u s i n g a Phillips Model E M - i o o A electron microscope.
Biochirn. Biophys. Acta, 55 (1962) 8 4 9 - 8 6 4
A.O. POGO, et al.
852
scribed previously 14. In this case, because Mg2+ions are essential to the preservation of ribosome structure, the sucrose isolation medium was modified, replacing the Ca *+ used in the original method with an equivalent amount of magnesium. The nuclear sediments were washed twice with 20 volumes of 0.25 M sucrose-o.oo3 M MgCI, and finally packed down b y centrifugation at IOOOx g for IO min.
Conditions o/ extraction The volume of the nuclear sediment was measured and the nuclei transferred to a stainless-steel blendor with a variable-speed motor and overhead-drive stirrer ("Omni-Mixer"; I v a n Sorvall, Inc. Norwalk, Conn.). Ten volumes of extracting medium were added and the nuclei disrupted b y homogenizing for I min at 8o00-9000 rev./min. Breakage of the nuclei is accompanied b y an increase in the viscosity of the suspension. This operation and all subsequent steps in the isolation procedure were carried out in the cold (0°-2°). Aliquots of the homogenate were taken for RNA, DNA and protein analyses. The ribonucleic acid content was determined b y the phloroglucinol method of DlSCHE AI~I~ BOREN~REONDTM. Deoxyribonucleic acid was determined b y the BVRTON modification 2° of the DlSCHE diphenylamine reaction ~1. Protein was estimated b y the Biuret reaction ~2. The homogenate was centrifuged at 30o0 × g for 30 min to remove nuclear debris and large aggregates. The extract was decanted and its volume measured. By comparing the RNA content of the extract with that of the original nuclear homogenate, an estimate could be made of the efficiency of extraction, i.e. RNAextraeteo/total RNA in homogenate. Using this method of homogenization, and the extraction media described below, up to 7 ° % of the total nuclear RNA could be extracted, while less than one per cent of the total DNA remains in the supernatant fraction after removal of the nuclear debris at 30o0 × g.
Influence o[ pH o/ the extraction medium The amount of RNA recovered in the nuclear extract varies depending upon the p H of the extracting medium. The relationship between p H and yield in Tris buffers is shown in Fig. 2. In this figure the data are summarized for two major conditions of extraction, termed "low" and "high" ionic strengths. In all experiments the Mg 2+ ion concentration was fixed at 5" lO-4 M. Fig. 2 A shows the results obtained at low ionic strengths using o.oi M Tris buffers in the p H range 7 to 9. The corresponding results obtained in o.I M Tris (high ionic strength) are plotted in Fig. 2 B. I n both cases the.yields increase with increasing pH. However, a study of the sedimentation properties of the ribosomes in these extracts shows that ribosome structure is disrupted to an increasing extent as the p H exceeds 8.0-8.5. For this reason all subsequent extractions were carried out at p H 7.6-7.7, despite the somewhat lower efficiency in terms of RNA recovery.
Influence o/ Mg 2+ concentration on ribosome yield Although some magnesium must be present in order to stabilize ribosome strucBiochim. Biophys. Acta, 55 (1962) 849-864
853
PURIFICATION OF NUCLEAR RIBOSOMES
A
B
0.01M "Tris"
0.1M "Tris"
7¢
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-~ 6c w .c .C_ 5C +
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~_Percent~of total DNA extracted
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7.0
I
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I
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9.0
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8.0
9,0
pH of extrocfincj buffer Fig. 2. Effect of increasing p H on the e x t r a c t i o n of ribosomes f r o m isolated t h y m u s nuclei. The p e r c e n t a g e of the t o t a l nuclear RNA, protein, or D N A which a p p e a r s in t h e e x t r a c t is plotted a g a i n s t the p H of the e x t r a c t i n g m e d i u m . Buffers used were o.oI M "Iris (A), and o.I M Tris (B). T h e Mg ~+ c o n c e n t r a t i o n w a s held c o n s t a n t at 5" IO-4 M.
ture 2s-25, excessive magnesium (or other divalent cation) concentrations reduce the yield in the nuclear extracts.The relationship between Mg ~+ concentration and RNA yield is shown in Fig. 3 A and 3 B. In these experiments the p H was kept constant at 7.7, and the concentration of MgC12 in the Tris buffer was varied as shown in the abscissae. At both "high" and "low" ionic strengths the yield of RNA in the nuclear extract begins to decrease after the Mg z+ concentration exceeds 5" lO-4 M. (It should be stressed, however, that the actual Mg 2+ concentration is higher than this because of the contribution of Mg 2+ originally present in the nucleus ae and ions adsorbed b y the nuclei in the course of their isolation ~6.
Results o[ extraction Although the yield of RNA in the nuclear extract varies somewhat from one preparation of nuclei to another, an average of 60-70 % of the total nuclear RNA can be recovered in extracts made with o.oi M Tris buffers (pH 7.6-7-7) containing 5" lO-4 M MgCI2, following the extraction procedure described above. The extractable RNA includes both the ribosomal RNA and the amino acid "transfer"-RNAs of the nucleus. Much of the non-extractable RNA is a component of the nucleoli s7 and is sedimented with the nuclear debris. Biochim. Biophys. Acta, 55 (1962) 849-864
854
A.O.
POCO,
et al.
A
0.1 M "Tris" (pH 7.7)
(3.01M "Tris" (pH 7.7) 80
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0.0o5
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Mg CIz Concentration (M) Fig. 3. Effect of increasing Mg ~+ concentration on the extraction of ribosomes from isolated t h y m u s nuclei. The percentage of the total nuclear RNA, protein, or D N A which appears in the e x t r a c t is indicated for different concentrations of MgC12. The p H was kept c o n s t a n t a t p H 7.7, using either o.oi M Tris-HC1 buffers (A), or o.I M Tris-HC1 (B).
Centri/ugation o/ ribosomes After removing the nuclear residues and heavy aggregates by centrifugation at 3000 x g for 3° rain, the Tris extracts are slightly opalescent. Large granules are then removed by centrifugation at 15 ooo x g for 15 min (in the No. 30 rotor of the SPINCO Model L preparative ultracentrifuge). This clarifies the extract considerably, with only slight losses of RNA; the sediment is discarded. Ribonucleoprotein particles have been collected from the supernatant fraction in two ways: in relatively small-scale preparations (i.e. from about 15 g of thymus nuclei (wet wt.)), the ribosomes are sedimented by centrifugation at lO5 ooo x g for 9 ° rain (using the No. 40 rotor of the SPINCO Model L). In large-scale preparations (employing 50-60 g of nuclei) the ribosomes are collected by centrifugation at 73 ooo X g for 18o min (using the No. 30 rotor). Examination of the ribonucleoprotein-containing pellets shows a dense aggregation of ribosomes together with other granules and adsorbed materials. Analysis of this crude ribosome fraction (Fraction I) gives 24.5+3.5 % RNA, 4.0±0.4 % DNA, and 71.3±3.o % protein. The "recoveries" in terms of RNA are high: 60-7o % of the total nuclear RNA is found in the "Iris extracts of thymus nuclei. About 85 % of this RNA sediments in the ribosome fraction; the remaining 15 % is discarded with the supernatant fraction, together with the soluble proteins of the nucleus. Biochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
855
Further stages in ribosome purification The ribosomes present in the crude ribosome fraction (Fraction I) can be further purified b y centrifugation in a sucrose gradient. The procedure used was that of BRITTEN AND ROBERTS ~s, modified for large scale isolation of ribosomes by using the SW 25 rotor of the SPINCO Model L ultracentrifuge. The sucrose density gradient was prepared b y mixing 14.2 ml of 5 % sucrose solution with 13.4 ml of 20 % sucrose. Both sucrose solutions contained 5" lO-4 M MgC12 and were buffered at p H 7.7 with o.ooi I M sodium phosphate. A mixing chamber similar to that described by BRITTEN AND ROBERTS~s was used. The crude ribosome pellet (Fraction i) was resuspended in o.ooli M sodium phosphate (pH 7.7)-0.0005 M MgCI~ at a concentration of 5-1o mg (dry wt.)/ml. This suspension was layered in an inverse concentration gradient over the sucrose gradient previously prepared, using 1.2 ml of ribosome suspension and 1.2 ml of 4 % sucrose solution in each arm of the mixing chamber ~s. The suspension was centrifuged at 60 ooo × g for 3 h. The centrifuge tube was then punctured at its base with a stainless-steel needle, and the drops were collected in a sequence of thirty I.o-ml fractions, o.I-ml aliquots of each fraction were diluted to I.O ml with water and the absorbancy measured at 235 m# and 260 m;u. The distribution of RNA, as indicated by the absorbancy readings, is shown in Fig. 4. In the peak, all fractions have an E280/E~5 ratio of 1.4 or higher. These fractions were pooled and the ribosomes precipitated by the addition of cold TCA to a final concentration of IO %. The precipitates were washed twice with cold 5 % TCA, ethanol, ethanol-ether (3:1) and ether, in that order. For analysis, the dried ribosomes were suspended in 5 % TCA and heated at 9 °o for 15 min to extract RNA.
2'°° 11.eol.eo20% '
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Fig. 4. Distribution of nuclear ribosomes after centrifugation in a sucrose density-gradient. Fractions were collected as described in the text and the ribosomes located b y their absorption a t 260 m/~. In this figure the absorbancy is plotted against fraction number, beginning with the denser fractions at the b o t t o m of the centrifuge tube and proceeding toward the lighter fractions at the top.
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A.O. POGO, et al.
The protein was centrifuged down. The RNA content of the extract was measured by the phloroglucinol reaction; the residue was dissolved in I N NaOH and its protein content determined by the Biuret reaction. The average composition of nuclear ribosomes purified in this way is shown in Table I. It can be seen that ribonucleic acid comprises at least 37 % of the mass of nuclear ribosomes purified in a gradient; the remainder is protein. It will be shown TABLE I COMPOSITION OF NUCLEAR RIBOSOME FRACTIONS AFTER DIFFERENT CONDITIONS OF ISOLATION
Material analyzed
Ionic strength
RNA
Percent of total mass as Protein
DNA
Number o] determinations
Cru de r i b o s o m e p e l l e t ( F r a c t i o n I) R i b o s o m e s p u r i f i e d in sucrose g r a d i e n t R i b o s o m e s t r e a t e d w i t h o.5 % d e o x y c h o l a t e Above ribosomes after treatment with o.ooi M E D T A
o.oi o.oi o.oi
24.5±3. 5 36.0 57.0±6.0
71.3±3.o 64.0 40.6±4.5
4.04-0-4 o o
3 2 5
o.oi
56.oi4.o
44.o~4.o
o
3
Crude r i b o s o m e p e l l e t ( F r a c t i o n I) R i b o s o m e s t r e a t e d w i t h 0.2 % L u b r o l Ribosomes treated with 0.2 % L u b r o l - o . 5 M KC1 R i b o s o m e s t r e a t e d w i t h 0.5 % L u b r o l R i b o s o m e s t r e a t e d w i t h 0. 5 % DOC R i b o s o m e s t r e a t e d w i t h I.O % DOC
o.I o.I
25. 5 36.0
72.0 63.0
2.5 2.0
3 2
o.5 o.I o.i o.I
43.0 34.0 62.o 62.0
57.o 65.0 38.o 38.0
i.o o o
--
2
i 3 3
below, and in a subsequent paper s, that much of this protein can be dissociated from the ribosomes by treatment with detergents. However, protein which can be dissociated from gradient-purified ribosomes by treatment with detergents is probably functionally associated with the ribosome, since kinetic studies of amino acid uptake show that this protein fraction is labeled more rapidly than the proteins in the soluble phase of the nucleus. No DNA has been detected in the ribosome peak obtained by centrifugation in a sucrose density gradient. The purified ribosome peak is composed largely of particles of sedimentation constant 78 S (at infinite dilution). The ultracentrifugal homogeneity of the ribonucleoprotein particles depends on the ionic strength and magnesium ion concentration of the medium, and the role of these variables in controlling ribosomal structure will be discussed in detail later in this paper. E//ect o/ detergents on nuclear ribosomes Detergents have been widely used in the purification of ribosomes from mammalian cytoplasm, and from bacterial and plant sources. Among those most commonly used are the non-ionic detergent, Lubrol W, and the ionic steroid, sodium deoxycholate. Lubrol W: This substance, a copolymer of polyoxyethylene and cetyl alcohol; made by Imperial Chemical Industries, Ltd., was used by COHN AND BUTLER in the purification of ribonucleoprotein particles from rat-liver microsomes .9. In the case of thymus nuclear ribosomes, the addition of Lubrol (at concentraBiochim.
Biophys.
Acta,
55 (1962) 8 4 9 - 8 6 4
PURIFICATION OF NUCLEAR RIBOSOMES
857
tions between 0.2 and 0.5 % (w/v)) to the crude ribosomal pellet (Fraction I) solubilizes about 4 ° % of the total protein and there is a concomitant rise in RNA content from 24. 5 to 35 % of the total weight. Even greater increases in RNA content of the ribosome pellet can be obtained if the ionic strength of the Lubrol solution is increased by the addition of neutral salts. This procedure was used by RENDI AND HULTIN in a purification of ribonucleoprotein particles from rat liver 3°. When thymus nuclear ribosomes were treated with 0. 5 % Lubrol in 0.5 M NaC1 and subsequently centrifuged at lO5 ooo × g for 2 h, the pellet was found to contain 43 % RNA and 57 % protein. Unfortunately, the addition of this much salt to the Lubrol solutions increases the ionic strength of the medium to an extent that causes dissociation of nuclear ribosomes, breaking the 78-S component into smaller units (see Fig. 5). For this
A CRUDE RIBOSOMES
--
115S 75S65S45S
D RIBOSOMES TREATED WITH DO(3
• Jl
C RIBOSOMES TREATED WITH NoCI
66S 40-55S
Fig. 5- U l t r a c e n t r i f u g a l p a t t e r n s of n u c l e a r r i b o s o m e fractions. N o t e t h e presence of a 75-s c o m p o n e n t , A, w h i c h b r e a k s d o w n w h e n r i b o s o m e s u s p e n s i o n s are t r e a t e d w i t h L u b r o l + N a C 1 , B, o. 5 M NaC1 alone, C, or w i t h d e o x y c h o l a t e a t ionic s t r e n g t h o.I, D. All p a t t e r n s o b t a i n e d a t 37 020 r e v . ] m i n ; A a n d D, a f t e r i o m i n a t 80°; B, a f t e r 18 rain a t 8°; C, a f t e r IO rain a t 25 °. I n p a t t e r n ]3, 64 S a n d 42 S c o r r e s p o n d to 75 S a n d 55 S w h e n m e a s u r e d in m o r e d i l u t e s u s p e n s i o n s . F o r f u r t h e r details see t e x t .
reason further purifications using Lubrol-salt combinations were not attempted, and we proceeded to test the effects of ionic detergents such as sodium deoxycholate, avoiding high salt concentrations. Sodium deoxycholate: The use of this substance in solubilizing the membrane fraction of rat-liver microsomes was first reported by STRITTMATTERAND BALL in their work on microsomal piglnents 31, and deoxycholate has been wide used elsewhere in the purification of the ribonucleoprotein particles of the cytoplasm32, sS. In tests on thymus nuclear ribosomes, it was found that much of the protein associated with the crude ribosomal pellet (Fraction I) can be solubilized b y treatment with sodium deoxycholate in the concentration range o.5-1.o %. Two conditions have been employed, depending on the ionic strength of the Tris buffer used in the initial extraction of the nuclei. Our first experiments were carried out at ionic strengths near o.I. The ribosomes were extracted in o.I M Tris buffers at p H 7.6 containing 5" lO-4 M MgC1v The crude ribosomal pellet (Fraction I) was resuspended in o.ooiI M sodium phosphate butter (pH 7.6) containing 5 . I o - 4 M MgCI~ and o.I/l~r KC1 (to maintain high ionic strength). The ribosome concentration of the susBiochim. Biophys. Acta, 55 (1962) 8 4 9 - 8 6 4
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A.O. POGO, et al.
pensions ranged from IO to 20 mg/ml. The suspensions were dialyzed against the buffer-KC1 mixture for 20 h at 2 °, and large particles were then removed by centrifugation at 15 ooo × g for 15 min. To the supernatant fraction, containing the ribosomes, was added a concentrated solution of sodium deoxycholate in buffer. (It is convenient, in carrying out this operation, to prepare a stock solution of 5 % DOC in water and to mix appropriate volumes of this solution with the buffer immediately before use. This avoids the formation of precipitates which occurs when deoxycholate and Mg~+ containing buffers are mixed and allowed to stand for prolonged periods.) In all our experiments an amount of deoxycholate was added to exceed the weight of ribosomes in the suspension by ten-fold. Nuclear ribosome suspensions became completely clear after standing for 20-30 min at 2 ° in the presence of deoxycholate. They were then centrifuged at lO5 ooo X g for 2 h. The pellet, consisting of purified ribosomes, occurs as a clear, firm gel. A small, overlying white sediment, which is not ribosomal, could be readily removed by washing the pellet with a small amount of buffer, swirling and decanting the white slurry. The clear pellet could then be transferred with the aid of a spatula to a glass homogenizing tube and then resuspended in the buffer using a slowly-rotating Teflon pestle. For purposes of analysis, aliquots of this suspension were added to equal volumes of 20 % trichloroacetic acid, and the precipitates treated with ethanol, ethanol-ether, and ether, as described previously. The composition of nuclear ribosomes treated with DOC in this way is given in Table I. Their high RNA content (62 %) is similar to that described for bacterial ribosomes by TISSIERES AND WATSON25, who analyzed the ribosomes of Escherichia coli and found them to contain 60-65 % RNA. The ribonucleic acid content of nuclear "DOC"-ribosomes is considerably higher than most figures reported for cytoplasmic ribosomes from other animal tissues. Guinea-pig liver ribosomes, for example, contain 40-45 % RNA, even though deoxycholate is used in the isolation ~. The increase in RNA content from 24.5 % in the crude ribosomal pellet (Fraction I) to over 60 % in nuclear "DOC"-ribosomes indicates that detergent treatment has removed much of the protein without concomitant losses in RNA content. Although the protein solubilized by deoxycholate is not all derived from ribosomes, part of it must have been associated with the ribosomes in a functional way. Thus the extract includes many amino acid "activating" enzymes. Moreover, tracer experiments using 14C labeled amino acids show that deoxycholate solubilizes many of the proteins synthesized by ribosomes which are still weakly associated with the synthetic site 5. Our first isolations of nuclear DOC-ribosomes were carried out at ionic strengths near o.I. It was soon found that the method required modification because examination of these preparations in the analytical ultracentrifuge showed the bulk of the material now appeared as a 48-S component. The relatively high ionic strength employed in the isolation had caused a dissociation of the 78-S component into the smaller units (see Fig. 5). To avoid this, subsequent isolations of nuclear ribosomes were carried out at much lower ionic strengths, extracting the ribosomes in o.oi M "iris buffers containg 5" lO-4 M MgCI~, and subsequently treating the crude ribosome pellet with 1 % deoxycholate in o.ooli M sodium phosphate-o.ooo5 M MgC1, (pH 7.6). Nuclear ribosomes prepared in the presence of deoxycholate at these low ionic Biochim. Biophys. dcta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
859
strengths occur largely as a 78-S component (Fig. 6). Their chemical composition is given in Table I, which presents the average of five different preparations under these conditions. Again it can be seen that RNA comprises about 6o % of the dry weight of TCA-washed ribosomes.
A DOG RIBOSOMES
103S ~ S S T S B UNTREATED RIBOSOMES
~PLUS EDTA
C ~lC
RIBOSOMES PLUS EDTA
,
t
Fig. 6. Ultracentrifugal patterns of nuclear ribosomes, showing effects of Mg~+ removal on ribosome structure. Note the presence of the 72-s component in purified ribosomes, A, which disappears when t~DTA is added, C. A similar effect is seen for crude ribosome suspensions {Compare (B) with Fig. 5 A). All patterns obtained at 37 o20 rev./min; A, after 14 min at 9°; B and C, after 18 rain at 8°. For further details see text. As in the case of cytoplasmic and bacterial ribosomes ~3-~s, the structure and sedimentation properties of the purified nuclear ribosomes depend to a large extent on the magnesium ion concentration of the suspending medium. At 5" lO-4 M Mg2+, the particles occur largely as a 78-S component. Removal of magnesium by dialysis against solutions of a chelating agent (e.g. EDTA) leads to an extensive disruption of the 78-s component, with the appearance of the smaller units of about 50 s, 42 s, and 28 s (Fig. 6). When EDTA-treated ribosomes are collected by centrifugation at lO5 ooo × g for 4 h, a pellet is obtained consisting of these smaller subunits. Analysis of the pellet shows the RNA content remains at about 60 % (Table I). Further details on the sedimentation properties of nuclear ribosomes will be presented below.
Nucleotide composition o/ ribosomal ribonucleic acid The average base composition of the RNA in nuclear ribosomes isolated with the aid of sodium deoxycholate has been determined. For this purpose 5-1o mg of ribosomes (dry wt.) were hydrolyzed in I N NaOH for 22 h at 30 °. The nucleotides were
Biochim. Biophys. Acta, 55 (1962) 849-864
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A.O. POGO, et al.
separated b y paper chromatography in the NH3-isobutyric acid solvent system of ELSON et al) s. The nucleotide composition of the ribosomal RNA is compared with that of the total nuclear RNA in Table II. It can be seen that the overall compositions are quite similar, and that ribosomal RNA has the usual high proportions of guanylic TABLE II NUCLEOTIDE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID FRACTIONS Moles]zoo moles total nucleotide present as Material analyzed
Adenylic acid
Uridylic acid
Total nuclear R N A " N u c l e o l a r " R N A fraction Nuclear ribosomal 1RNA Nuclear " M e s s e n g e r " R N A
20.4 21.8 20. 4 29.3
19.3 23.5 18.6 28.4
33.3 3o.5 32.7 22.5
27.8 25.7 28.2 19.8
Thymidylic 28. 3
22.6
19.9
T h y m u s DNA*
27.6
Guanylie acid
Cytidylic acid
Relerence
(27) (36)
* F o r p u r p o s e s of comparison.
and cytidylic acids. It should be stressed however, that not all nuclear RNAs have the same base ratios, and that thymus nuclear RNA includes a small but metabolically very active fraction, which is low in guanylic and cytidylic acids, and high in adenylic and uridylic acids, and thus resembles thymus DNA in its base composition (if one considers RNA-uridylic acid equivalent to DNA thymidylic acidae). Electron microscopy o[ nuclear ribosomes Ribosomes isolated in the presence of sodium deoxycholate have been examined under the electron microscope. In preparing these ribosomes for electron microscopy, the DOC-ribosome pellet was resuspended in o.ooli M sodium phosphate-o.ooo5 M MgClz at pH 7.6. Droplets of the suspension were transferred directly to "formvar" films on copper grids. The liquid phase was rapidly absorbed by sintered glass discs placed beneath the grids. The ribosomes on the film were further washed free of sucrose by washing three times with o.I-ml portions of the magnesium-containing buffer. After drying, the films were shadow-cast with chromium at an angle of 15 ° . The pictures were taken in the Hitachi Model H U - I I electron microscope at plate magnifications of 20 ooox and 40 ooox. In one experiment, the DOC-ribosomes were further purified by centrifugation in a sucrose density-gradient, as described earlier. After locating the ribosome peak by its absorption at 260 m/~, o. I-ml aliquots of the peak solution were transferred to "formvar" films on copper grids. The ribosomes were washed and shadow-cast with chromium, as described above. The results are shown in Fig. 7, A and t3. It can be seen that nuclear ribosomes are roughly spherical or ellipsoidal and that several size classes are represented in the pictures. When dimensions are measured perpendicular to the direction of shadowing, it is found that most of the particles are about 260/~. units wide, but others range in size from 13o A to 350 A. The 26o-A particles are considered to represent Biochim.
Biophys.
Acta,
55 (1962) 8 4 9 - 8 6 4
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the 78-s ribosomes, since the latter comprise the most prevalent component in ultracentrifugal analysis. The dimension 260 A includes the cap of chromium metal deposited by shadowcasting; this exaggerates the apparent width of the particle, as was pointed out for E. coli ribosomes by HALL AND SLAYTER37. They found that shadow-casting with
Fig. 7. E l e c t r o n m i c r o g r a p h s of t h y m u s n u c l e a r ribosomes. T h e r i b o s o m e s were p r e p a r e d w i t h d e o x y c h o l a t e a t low ionic s t r e n g t h s a n d t h e n purified in a sucrose d e n s i t y - g r a d i e n t . D r o p l e t s of t h e purified r i b o s o m e s u s p e n s i o n were dried on " F o r m v a r " f i l m s a n d s h a d o w e d w i t h c h r o m i u m a t a n a n g l e of 15 ° . F o r f u r t h e r details see t e x t .
platimum increased the dimensions of E. coli ribosomes by about 60 A. If a similar correction is made in the case of thymus nuclear ribosomes, the corrected width is about 200/~. (This dimension is still considerably higher than the figures previously reported for nuclear ribosomes fixed in situ with osmium tetroxidO; osmium-fixed ribosomes are usually lOO-15o/~ in diameter. The discrepancy is probably due to the great differences in fixation method and preparation of samples.) It is of interest that both thymus nuclear ribosomes (78 s) and E. coli 7o s ribosomes are about 200 A across, and that both contain the same relative proportions of RNA and total protein. Ultracentri/ugal analysis o/ thymus nuclear ribosomes Before analysis, nuclear ribosomes were dialyzed overnight at 2° against buffer systems commonly employed in the study of cytoplasmic ribosomes. Two systems were used (a) o.I M KCl-o.oolI M potassium phosphate, pH 7.4-o.ool 5 M MgCI~, and (b) o.ooli M potassium phosphate, pH 7.4, containing 0.0005 M MgCI~; this Biochim. Biophys. Acfa, 55 (I962) 8 4 9 - 8 6 4
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A.O. POGO, et al.
was made up to o.o3 M in KC1 just before analysis. These systems will be referred to as "o.I" and "0.03" ionic strength buffers. The samples were centrifuged in 12 ram, 2 ° double-sector cells at 37 020 rev./min, and, unless otherwise indicated, analyses were performed at 8 °. The schlieren pictures were enlarged and traced. Sedimentation coefficients were calculated in the usual way and the concentrations were computed from the areas under the curves, assuming a specific refractive index increment of o.oo187 for a I °/o solution. Analysis of the crude ribosomal pellet (Fraction I) shows that it consists of a mixture of particles of differing sedimentation properties. At ionic strengths near o.I the schlieren pattern consistently showed 4 or 5 ribosome peaks as well as a large, slowly-sedimenting boundary (Fig. 5 A). The chief component (which accounted for approx. 4 ° ~o of the total pattern area) had a sedimentation constant of about 75 S (uncorrected) at a total concentration of 4 mg/ml. The other components had sedimentation constants of 115 S, 65 S, and 45 S. This pattern is similar to that obtained for ribosomes from a variety of other sources (see ref. 38). In addition to the ribosome peaks, smaller components with sedimenta[ion constants near IO s, and occasionally a 2o-S component were observed; these accounted for about 30 % of the total area of the pattern. (A 2o-S component has also been found b y PETERMANN in unpurified Jensen sarcoma ribosomes but was shown to be lacking in ribonucleic acid~9).
E][ects o/detergents and salts on nuclear ribosome structure Lubrol-NaCl: Examination of ribosome suspensions prepared with the aid of Lubrol-salt solutions showed evidence of extensive dissociation of the particles. Although essentially the same components were observed as in untreated ribosomes, their relative proportions had changed considerably (Fig. 5 B). The 75-S ribosomes were present in much lower concentration than in untreated samples; instead a 55-S peak began to predominate in the schheren pattern. The II5-S component was not a distinct peak but a mixture of polydisperse materials. Also present was IO S material, in approximately the same percentage as in untreated ribosome suspensions. Attempts to reverse the dissociation by doubling the magnesium ion concentration (see below) had only a small effect, augmenting the 75-S component and decreasing the 55-S component shghtly. (Similarly, the addition of spermidine phosphate to a final concentration of 0.005 M removed the 55-S peak and led to the appearance of 69-7I-S components and some polydisperse heavy material.) Since the Lubrol treatment is carried out in the presence of 0.5 M NaCI, the effect of salt alone was tested, with the results shown in Fig. 5 C. The pattern shows almost complete conversion of the 75-S ribosomes to material with sedimentation values between 33 S and 40 S, and a sizeable increase in the percentage of Io-S material This it seems clear that the Lubrol+salt treatment is damaging. Sodium deoxycholate: A similar examination of the effects of deoxycholate on the nuclear ribosomes revealed a marked dependence on the ionic strength of the medium. Ribosomes prepared with DOC at ionic strengths near o.I gave the pattern shown in Fig. 5 D. Although a 74-S component was present, the bulk of the pattern was made up of a 48-S component. (As in the case of Lubrol-NaC1 treated ribosomes,'the addition of spermidine phosphate to these DOC ribosomes seemed to convert the 48-S Biochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
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material to larger units, thus increasing the 74-S peak and the fast polydisperse boundary. ) When the ribosomes were extracted from the nuclei in very dilute Tris buffers (o.oi M) and subsequently treated with deoxycholate at low ionic strengths, much less dissociation was observed. Under these conditions of low ionic strength, 7° ~ of the pattern consisted of a 72-S component; in addition there was 15 % of a Io3-S component and 15 % of 57-S material (Fig. 6 A). No slowly sedimenting material was observed. The sedimentation coeHicient o[ nuclear DOC-ribosomes Ribosomes prepared with the aid of deoxycholate at low ionic strengths were analyzed in the ultracentrifuge at three concentrations, 3.1, 1.2, and 0. 9 mg/ml (keeping the ionic strength constant at 0.03). Schlieren optics were used. The sedimantation coefficients, s20,w, for the three analyses were 71.8, 74.2 and 74.6. These extrapolated to a value of s20,w, of 75.6- Thus nuclear ribosomes resemble other ribosomes in the strong concentration dependence of their sedimentation rates. The DOC-ribosomes were also examined with ultraviolet optics at a concentration close to o.05 mg/ml. The sedimentation coefficient in this case was 77-9 S. Thus, with a sedimentation coefficient at infinite dilution of about 78 S, nuclear ribosomes fall into the rather large class of 8o-S ribosomes. Role o[ Mg 2+ ions in nuclear ribosome structure All ribosomes seem to be composed of nucleoprotein subunits held together by magnesium ions. The role of Mg2+ in nuclear ribosome structure was made evident in experiments in which the chelating agent, EDTA, was added. In untreated ribosome suspensions, in which the 74-S component predominates, small amounts of EDTA led to the appearance of 64-S and 42-S components; at o.oi M EDTA only 49-S and 33-S components were present (Fig. 6 B). In the deoxycholate-treated ribosomes prepared at low ionic strengths, EDTA acted in a similar fashion, disrupting the 72-74 S component completely to units of 42 S and 28 S (compare Figs. 6 C and 6 D). These results resemble those obtained by PETERMANN AND HAMILTONwith ratliver ribosomes88, where the 83-S component breaks down into particles of 46 S and 30 S when the Mg2+ concentration is lowered. Thus, the 78-S nuclear ribosome, like many other ribosomes, appears to be an aggregate of smaller subunits, and magnesium ions play a role in this aggregation.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. M. L. PETERMANN of the Sloan-Kettering Institute for her advice and for many helpful discussions. This research was supported in part by a Grant (RG 4919) from the U.S. Pubhc Health Service. The first Author is a Fellow of the Guggenheim Foundation, and B. G. T. P. a Fellow of the Consejo Nacional de Investigaciones Cientlficas y T6cnicas, of the Argentine Republic. V. C. L. and M. G. H. were supported by the United States Atomic Energy Commission, the latter under their Contract AT(3o-I)-9io. Biochim. Biophys. Acta, 55 (I962) 849-864
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A.O. POGO, et al. REFERENCES
' J. W. HOPKINS, Proc, Natl. Acad. Sci. U.S., 45 (1959) 1461. 2 V. G. ALLEREY, J. W. HOPKINS, J. H. FRENSTER AND A. E. MIRSKY, Ann. N . Y . Acad. Sci., 88 (196o) 722. a j . W . HOPKINS, V. G. ALLFREY AND A. E. MIRSKY, Bioehim. Biophys. Acta, 47 (1961) 195. 4 j. H. FRENSTER, V. G. ALLFREY AND A. E. 1VilRSKY, Proc. Natl. Acad. Sei. U.S., 46 (196o) 432. 5 V. G. ALLFREY, A. O. POGO, B. G. T. PoGo AND A. E. ~V[IRSKY, m a n u s c r i p t in p r e p a r a t i o n . 6 K. R. PORTER, J. Histochem. Cytochem., 2 (1954) 346. 7 W. BERNHARD, A. BAUER, A. GROPP, F. HAGENAU AND C. OBERLING, Exptl. Cell Kesearch, 9 (1955) 88. s j . G. LAFONTAINE, J. Biophys. Biochem. Cytol., 4 (1958) 229. 9 j . G. GALL, J. Biophys. Biochem. Cytol. Suppl. 2 (1956) 4o7 . 10 S. WlSCHNI~ZER, J. Ultrastruct. Research, I (1958) 2Ol. n M. L. WATSON, J. Biophys. Biochem. Cytol., 6 (1959) 147. 1, W. BEERMAN AND G. F. BAHR, Exptl. Cell Research, 6 (1954) 195. 13 H. G. CALLAN, Symposium on Fine Structure o/ Cells (Leiden), I n t e r n . Union Biol. Sci. Publ. B2I (1956 ) 89. 14 V. G. ALLFREY, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1957) 451. 15 T. Y. WANG, Biochim. Biophys. Acta, 45 (196o) 8. x8 V. G. ALLFREY, R. MEUDT, J. W. HOPKINS AND A. E, MIRSK¥, Proc. Natl. Acad. Sei. U.S., 47 (1961) 9o7 . x7 H. SACHS, J. Biol. Chem., 228 (1957) 23. 18 V. G. ALLFREY, M. M. DALY AND A. E. MIRSKY, J. Gen. Physiol., 37 (1953) 157. IS Z. DISCHE AND E. BORENFREUND, Biochim. Biophys. Acta, 23 (1957) 639. s0 K. BURTON, Biochem. J., 62 (1956) 315 • 21 Z, DlSCHE, Mikrochemie, 8 (193 o) 4. 22 C. F. CRAMPTON, R. LIPSHITZ AND E. CHARGAFF,J. Biol. Chem., 206 (1954) 499. ,3 p, O. P. T ' s o , J. BONNER AND J. VINOGRAD, Biochim. Biophys. ~'tcta, 3 ° (1958) 57 o. 38 M. L. PETERMANN, J. Biol. Chem., 235 (196o) 1998. ,s A. TlSSI~RES AND J. D. WATSON, Nature, 182 (1958) 778. 26 l-I. ~AORA, H. NAORA, A. E. MIRSKY AND V. G. ALLFREY, J. Gen. Physiol., 44 (1961) 713 • ,7 V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Aead. Sei. U.S., 43 (1957) 821. is R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. 38 p, COHN AND J. A. V. BUTLER, Biochem. J., 7 ° (1958) 254. 30 R. RENDI AND T. HULTIN, Exptl. Cell Research, 18 (1959) 542. 31 C. F. STRITTMATTER AND E. G. BALL, Proc. Natl. Acad. Soc. U.S., 38 (1952) 19. 32 j . W. LITTLEFIELD, E. B. KELLER, J. GRoss AND P. C. ZAMECNIK, J. Biol. Chem., 217 (1955) 11. 3a p. O. P. T s ' o AND J. VINOGRAD, Biochim. Biophys. Acta, 49 (1961) 113. 34 j . F. KIRSCH, P. SIEKEVlTZ AND G. E. PALADE, J. Biol. Chem., 235 (196o) 1419. a3 D. ELSON, T. GUSTAFSON AND E. CHARGAEF, J. Biol. Chem., 209 (1954) 285. 3s A. SIBATANI, A. E. ~/[IRSKYAND V. G. ALLFREY, Proc. Natl. Acad. Sci. U.S., in t h e press. 37 C. E. HALL AND H. S. SLAYTER, J. Mol. Biol., i (1959) 329. 3s ~VI.L. PETERMANN AND M. G. HAMILTON, in R. J. C. HARRIS, Physicochemical Studies on Ribonueleoproteins [rom Mammalian Cytoplasm, in Protein Biosynthesis, L o n d o n Academic Press, Ltd., 1961, p. 233. 3, IV[. L. PETERMANN, personal c o m m u n i c a t i o n .
Biochim. Biophys. Acta, 55 (1962) 849-864
840
THE PURIFICATION AND PROPERTIES OF RIBOSOMES FROM THE THYMUS NUCLEUS A. O. POGO*, B. G. T. POGO, V. C. LITTAU, V. G. ALLFREY AND A. E. MIRSKY The Rocke[eller Institute, New York, N . Y . (U.S.A.) AND
M. G. HAMILTON The Sloan-Kettering Institute New York, N . Y , (U.S.A.)
(Received November 27th, 1951)
SUMMARY Methods are described for the isolation and purification of ribonucleoprotein particles (ribosomes) from isolated calf-thymus nuclei. About half of the total ribonucleic acid of the nucleus can be recovered in the purified ribosome fraction. Nuclear ribosomes can be purified by centrifugation in a sucrose density-gradient. The ribosome peak contains about 36 % RNA. Treatment of the peak material with deoxycholate removes much of the protein. The resulting ribosomes have a sedimentation coefficient of 78 S and about 6o % of their mass is RNA. The average base composition of this ribosomal RNA is given and compared with that of other nuclear RNA fractions. The changes in state of the nuclear 78-S ribosome with changing pH, ionic strength, and Mg2+ concentration are described. Electron microscopy of isolated ribosomes after shadow-casting with chromium indicates that the 78-S ribosomes are about 200 A in width.
INTRODUCTION Previous studies of the mechanism of protein synthesis in isolated thymus nuclei have shown that amino acid incorporation into nuclear proteins involves the participation of ATP and amino acid "activating" enzymes1, 2, "transfer"-ribonucleic acidsZ, 3, and ribonucleoprotein particles, or ribosomes 2,*. The ribosome fractions prepared from isolated cell nuclei by differential centrifugation have been found to comprise a heterogeneous mixture of particles of differing composition, sedimentation properties, and metabolic activity 4. It is the aim of the present paper to (a) describe the extraction conditions leading to optimal yields and preservation of structure of thymus-nuclear ribosomes, (b) to present methods for the purification of nuclear ribosomes by centrifugation in a Abbreviations: RNP, ribonucleoprotein particles; TCA, trichloroacetic acid; DOC, deoxycholate. * Present address: Instituto de Biologia Cellular, Universidad Nacional de Cordoba, Casilla Correo 362, Cordoba (Argentina). Biochim. Biophys. Acta, 55 (1962) 849-864
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sucrose density gradient, (c) to summarize studies of the effects of detergents (Lubrol W and sodium deoxyholate) on ribosome structure, and (d) to present the composition and sedimentation properties of highly purified nuclear ribosome fractions, as well as their appearance under the electron microscope. In a subsequent paper s, the incorporation of [14Clamino acids into different components of these purified ribosomes will be described, together with kinetic evidence that protein synthesized in nuclear ribosomes acts as precursor to the proteins in the soluble phase of the nucleus.
Extraction o/ nuclear ribosomes The presence of granules lOO-300 A in diameter within the thymocyte nucleus is evident in electron micrographs of thymus tissue sections 4. The granularity of the nucleus is evident even at relatively low magnifications (Fig. I). Particles of similar magnitude have been observed previously in nucleoli 6-s, on the lateral loops of lampbrush chromosomes 9, in association with nuclear membranes1°, 11, on the chromosomal rings of BALBIANITM, and distributed throughout the nuclear sap4, ~S. When isolated thymus nuclei are extracted with neutral buffer or saline solutions the particles are removed together with other proteins of the soluble phase of the nucleus4,14,~5; the particles can then be recovered from the extract b y centrifugation at high speeds. They have been found to contain a large proportion of ribonucleic acid4,15. The evidence that ribonucleoprotein particles so prepared are indeed derived from the nucleus is based not only on electron microscopy before and after extraction 4, but also on chemical and morphological evidence for the purity of the nuclear preparations employed in their isolation, and on functional tests which indicate that RNP isolated after labeling experiments with 14C amino acids must have been present inside the nucleus at the time of ~ac uptake. For example, it has been shown that thymus-nuclear ribosomes require a sodium environment for their activity in situa, ~6, whereas free cytoplasmic ribosomes are strongly dependent on potassium ions ~. Therefore, amino acid incorporation observed in a potassium-poor, sodium-rich medium is not likely to be due to cytoplasmic ribosomes adsorbed on the outer membrane of the isolated nucleus. Moreover, protein synthesis by the ribosomes of the nucleus ceases when the nuclei are treated with deoxyribonuclease, but not when ribonuclease is added to the medium4,14. Since intranuclear ribosomes are not susceptible to attack by ribonuclease as long as they remain inside the nucleus, while extranuclear or cytoplasmic ribosomes are readily disrupted by pancreatic RNAase4, is this enzyme effect allows an unequivocal test for the presence and activity of ribosomes within the cell nucleus. The extraction of nuclear ribosomes was originally carried out in 0.I M phosphate buffer ~4 or Tris buffers at p H 7.1 (see ref. 4). Subsequent work indicates that the pH, ionic strength, and Mg 2+ ion concentration of the extracting medium all affect the state and recovery of nuclear RNP particles. These variables were then investigated systematically with a view to selecting conditions which allow a maximum yield and optimal preservation of structure of ribosomes in the nuclear extract.
Isolation o/ nuclei Nuclei were isolated from fresh calf thymus tissue following the methods deBiochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
85I
Fig. I. E l e c t r o n m i c r o g r a p h of t h y m u s cells in t i s s u e section. T h e sections were fixed for i h in b u f f e r e d o s m i u m tetroxide, e m b e d d e d in m e t h a c r y l a t e a n d s t a i n e d w i t h u r a n y l acetate. N o t e t h e s m a l l e l e c t r o n - d e n s e g r a n u l e s w i t h i n t h e nuclei; t h e s e d i s a p p e a r w h e n t h e n u c l e a r r i b o s o m e s a r e e x t r a c t e d in Tris b u f f e r s as described in t h e t e x t . T h i s electron m i c r o g r a p h w a s t a k e n b y B. S. McEWEN of t h e Rockefeller I n s t i t u t e u s i n g a Phillips Model E M - i o o A electron microscope.
Biochirn. Biophys. Acta, 55 (1962) 8 4 9 - 8 6 4
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scribed previously 14. In this case, because Mg2+ions are essential to the preservation of ribosome structure, the sucrose isolation medium was modified, replacing the Ca *+ used in the original method with an equivalent amount of magnesium. The nuclear sediments were washed twice with 20 volumes of 0.25 M sucrose-o.oo3 M MgCI, and finally packed down b y centrifugation at IOOOx g for IO min.
Conditions o/ extraction The volume of the nuclear sediment was measured and the nuclei transferred to a stainless-steel blendor with a variable-speed motor and overhead-drive stirrer ("Omni-Mixer"; I v a n Sorvall, Inc. Norwalk, Conn.). Ten volumes of extracting medium were added and the nuclei disrupted b y homogenizing for I min at 8o00-9000 rev./min. Breakage of the nuclei is accompanied b y an increase in the viscosity of the suspension. This operation and all subsequent steps in the isolation procedure were carried out in the cold (0°-2°). Aliquots of the homogenate were taken for RNA, DNA and protein analyses. The ribonucleic acid content was determined b y the phloroglucinol method of DlSCHE AI~I~ BOREN~REONDTM. Deoxyribonucleic acid was determined b y the BVRTON modification 2° of the DlSCHE diphenylamine reaction ~1. Protein was estimated b y the Biuret reaction ~2. The homogenate was centrifuged at 30o0 × g for 30 min to remove nuclear debris and large aggregates. The extract was decanted and its volume measured. By comparing the RNA content of the extract with that of the original nuclear homogenate, an estimate could be made of the efficiency of extraction, i.e. RNAextraeteo/total RNA in homogenate. Using this method of homogenization, and the extraction media described below, up to 7 ° % of the total nuclear RNA could be extracted, while less than one per cent of the total DNA remains in the supernatant fraction after removal of the nuclear debris at 30o0 × g.
Influence o[ pH o/ the extraction medium The amount of RNA recovered in the nuclear extract varies depending upon the p H of the extracting medium. The relationship between p H and yield in Tris buffers is shown in Fig. 2. In this figure the data are summarized for two major conditions of extraction, termed "low" and "high" ionic strengths. In all experiments the Mg 2+ ion concentration was fixed at 5" lO-4 M. Fig. 2 A shows the results obtained at low ionic strengths using o.oi M Tris buffers in the p H range 7 to 9. The corresponding results obtained in o.I M Tris (high ionic strength) are plotted in Fig. 2 B. I n both cases the.yields increase with increasing pH. However, a study of the sedimentation properties of the ribosomes in these extracts shows that ribosome structure is disrupted to an increasing extent as the p H exceeds 8.0-8.5. For this reason all subsequent extractions were carried out at p H 7.6-7.7, despite the somewhat lower efficiency in terms of RNA recovery.
Influence o/ Mg 2+ concentration on ribosome yield Although some magnesium must be present in order to stabilize ribosome strucBiochim. Biophys. Acta, 55 (1962) 849-864
853
PURIFICATION OF NUCLEAR RIBOSOMES
A
B
0.01M "Tris"
0.1M "Tris"
7¢
o
-~ 6c w .c .C_ 5C +
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~_Percent~of total DNA extracted
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o
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~
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I
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I
8.0
I
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9,0
pH of extrocfincj buffer Fig. 2. Effect of increasing p H on the e x t r a c t i o n of ribosomes f r o m isolated t h y m u s nuclei. The p e r c e n t a g e of the t o t a l nuclear RNA, protein, or D N A which a p p e a r s in t h e e x t r a c t is plotted a g a i n s t the p H of the e x t r a c t i n g m e d i u m . Buffers used were o.oI M "Iris (A), and o.I M Tris (B). T h e Mg ~+ c o n c e n t r a t i o n w a s held c o n s t a n t at 5" IO-4 M.
ture 2s-25, excessive magnesium (or other divalent cation) concentrations reduce the yield in the nuclear extracts.The relationship between Mg ~+ concentration and RNA yield is shown in Fig. 3 A and 3 B. In these experiments the p H was kept constant at 7.7, and the concentration of MgC12 in the Tris buffer was varied as shown in the abscissae. At both "high" and "low" ionic strengths the yield of RNA in the nuclear extract begins to decrease after the Mg z+ concentration exceeds 5" lO-4 M. (It should be stressed, however, that the actual Mg 2+ concentration is higher than this because of the contribution of Mg 2+ originally present in the nucleus ae and ions adsorbed b y the nuclei in the course of their isolation ~6.
Results o[ extraction Although the yield of RNA in the nuclear extract varies somewhat from one preparation of nuclei to another, an average of 60-70 % of the total nuclear RNA can be recovered in extracts made with o.oi M Tris buffers (pH 7.6-7-7) containing 5" lO-4 M MgCI2, following the extraction procedure described above. The extractable RNA includes both the ribosomal RNA and the amino acid "transfer"-RNAs of the nucleus. Much of the non-extractable RNA is a component of the nucleoli s7 and is sedimented with the nuclear debris. Biochim. Biophys. Acta, 55 (1962) 849-864
854
A.O.
POCO,
et al.
A
0.1 M "Tris" (pH 7.7)
(3.01M "Tris" (pH 7.7) 80
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Mg CIz Concentration (M) Fig. 3. Effect of increasing Mg ~+ concentration on the extraction of ribosomes from isolated t h y m u s nuclei. The percentage of the total nuclear RNA, protein, or D N A which appears in the e x t r a c t is indicated for different concentrations of MgC12. The p H was kept c o n s t a n t a t p H 7.7, using either o.oi M Tris-HC1 buffers (A), or o.I M Tris-HC1 (B).
Centri/ugation o/ ribosomes After removing the nuclear residues and heavy aggregates by centrifugation at 3000 x g for 3° rain, the Tris extracts are slightly opalescent. Large granules are then removed by centrifugation at 15 ooo x g for 15 min (in the No. 30 rotor of the SPINCO Model L preparative ultracentrifuge). This clarifies the extract considerably, with only slight losses of RNA; the sediment is discarded. Ribonucleoprotein particles have been collected from the supernatant fraction in two ways: in relatively small-scale preparations (i.e. from about 15 g of thymus nuclei (wet wt.)), the ribosomes are sedimented by centrifugation at lO5 ooo x g for 9 ° rain (using the No. 40 rotor of the SPINCO Model L). In large-scale preparations (employing 50-60 g of nuclei) the ribosomes are collected by centrifugation at 73 ooo X g for 18o min (using the No. 30 rotor). Examination of the ribonucleoprotein-containing pellets shows a dense aggregation of ribosomes together with other granules and adsorbed materials. Analysis of this crude ribosome fraction (Fraction I) gives 24.5+3.5 % RNA, 4.0±0.4 % DNA, and 71.3±3.o % protein. The "recoveries" in terms of RNA are high: 60-7o % of the total nuclear RNA is found in the "Iris extracts of thymus nuclei. About 85 % of this RNA sediments in the ribosome fraction; the remaining 15 % is discarded with the supernatant fraction, together with the soluble proteins of the nucleus. Biochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
855
Further stages in ribosome purification The ribosomes present in the crude ribosome fraction (Fraction I) can be further purified b y centrifugation in a sucrose gradient. The procedure used was that of BRITTEN AND ROBERTS ~s, modified for large scale isolation of ribosomes by using the SW 25 rotor of the SPINCO Model L ultracentrifuge. The sucrose density gradient was prepared b y mixing 14.2 ml of 5 % sucrose solution with 13.4 ml of 20 % sucrose. Both sucrose solutions contained 5" lO-4 M MgC12 and were buffered at p H 7.7 with o.ooi I M sodium phosphate. A mixing chamber similar to that described by BRITTEN AND ROBERTS~s was used. The crude ribosome pellet (Fraction i) was resuspended in o.ooli M sodium phosphate (pH 7.7)-0.0005 M MgCI~ at a concentration of 5-1o mg (dry wt.)/ml. This suspension was layered in an inverse concentration gradient over the sucrose gradient previously prepared, using 1.2 ml of ribosome suspension and 1.2 ml of 4 % sucrose solution in each arm of the mixing chamber ~s. The suspension was centrifuged at 60 ooo × g for 3 h. The centrifuge tube was then punctured at its base with a stainless-steel needle, and the drops were collected in a sequence of thirty I.o-ml fractions, o.I-ml aliquots of each fraction were diluted to I.O ml with water and the absorbancy measured at 235 m# and 260 m;u. The distribution of RNA, as indicated by the absorbancy readings, is shown in Fig. 4. In the peak, all fractions have an E280/E~5 ratio of 1.4 or higher. These fractions were pooled and the ribosomes precipitated by the addition of cold TCA to a final concentration of IO %. The precipitates were washed twice with cold 5 % TCA, ethanol, ethanol-ether (3:1) and ether, in that order. For analysis, the dried ribosomes were suspended in 5 % TCA and heated at 9 °o for 15 min to extract RNA.
2'°° 11.eol.eo20% '
~
gradient
, 5%
120
>-
~
t.00
~
o.8o
0:eo
'
2
o.4o
o.20
0
II
t]
~I1~
IltjIir
It I I I I I r t3f III L~ 17 b9 ~l I ~I t ~I I 27 2~ Fraction Number
Fig. 4. Distribution of nuclear ribosomes after centrifugation in a sucrose density-gradient. Fractions were collected as described in the text and the ribosomes located b y their absorption a t 260 m/~. In this figure the absorbancy is plotted against fraction number, beginning with the denser fractions at the b o t t o m of the centrifuge tube and proceeding toward the lighter fractions at the top.
Biochim. Biophys. Acta, 55 (1962) 849-864
856
A.O. POGO, et al.
The protein was centrifuged down. The RNA content of the extract was measured by the phloroglucinol reaction; the residue was dissolved in I N NaOH and its protein content determined by the Biuret reaction. The average composition of nuclear ribosomes purified in this way is shown in Table I. It can be seen that ribonucleic acid comprises at least 37 % of the mass of nuclear ribosomes purified in a gradient; the remainder is protein. It will be shown TABLE I COMPOSITION OF NUCLEAR RIBOSOME FRACTIONS AFTER DIFFERENT CONDITIONS OF ISOLATION
Material analyzed
Ionic strength
RNA
Percent of total mass as Protein
DNA
Number o] determinations
Cru de r i b o s o m e p e l l e t ( F r a c t i o n I) R i b o s o m e s p u r i f i e d in sucrose g r a d i e n t R i b o s o m e s t r e a t e d w i t h o.5 % d e o x y c h o l a t e Above ribosomes after treatment with o.ooi M E D T A
o.oi o.oi o.oi
24.5±3. 5 36.0 57.0±6.0
71.3±3.o 64.0 40.6±4.5
4.04-0-4 o o
3 2 5
o.oi
56.oi4.o
44.o~4.o
o
3
Crude r i b o s o m e p e l l e t ( F r a c t i o n I) R i b o s o m e s t r e a t e d w i t h 0.2 % L u b r o l Ribosomes treated with 0.2 % L u b r o l - o . 5 M KC1 R i b o s o m e s t r e a t e d w i t h 0.5 % L u b r o l R i b o s o m e s t r e a t e d w i t h 0. 5 % DOC R i b o s o m e s t r e a t e d w i t h I.O % DOC
o.I o.I
25. 5 36.0
72.0 63.0
2.5 2.0
3 2
o.5 o.I o.i o.I
43.0 34.0 62.o 62.0
57.o 65.0 38.o 38.0
i.o o o
--
2
i 3 3
below, and in a subsequent paper s, that much of this protein can be dissociated from the ribosomes by treatment with detergents. However, protein which can be dissociated from gradient-purified ribosomes by treatment with detergents is probably functionally associated with the ribosome, since kinetic studies of amino acid uptake show that this protein fraction is labeled more rapidly than the proteins in the soluble phase of the nucleus. No DNA has been detected in the ribosome peak obtained by centrifugation in a sucrose density gradient. The purified ribosome peak is composed largely of particles of sedimentation constant 78 S (at infinite dilution). The ultracentrifugal homogeneity of the ribonucleoprotein particles depends on the ionic strength and magnesium ion concentration of the medium, and the role of these variables in controlling ribosomal structure will be discussed in detail later in this paper. E//ect o/ detergents on nuclear ribosomes Detergents have been widely used in the purification of ribosomes from mammalian cytoplasm, and from bacterial and plant sources. Among those most commonly used are the non-ionic detergent, Lubrol W, and the ionic steroid, sodium deoxycholate. Lubrol W: This substance, a copolymer of polyoxyethylene and cetyl alcohol; made by Imperial Chemical Industries, Ltd., was used by COHN AND BUTLER in the purification of ribonucleoprotein particles from rat-liver microsomes .9. In the case of thymus nuclear ribosomes, the addition of Lubrol (at concentraBiochim.
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tions between 0.2 and 0.5 % (w/v)) to the crude ribosomal pellet (Fraction I) solubilizes about 4 ° % of the total protein and there is a concomitant rise in RNA content from 24. 5 to 35 % of the total weight. Even greater increases in RNA content of the ribosome pellet can be obtained if the ionic strength of the Lubrol solution is increased by the addition of neutral salts. This procedure was used by RENDI AND HULTIN in a purification of ribonucleoprotein particles from rat liver 3°. When thymus nuclear ribosomes were treated with 0. 5 % Lubrol in 0.5 M NaC1 and subsequently centrifuged at lO5 ooo × g for 2 h, the pellet was found to contain 43 % RNA and 57 % protein. Unfortunately, the addition of this much salt to the Lubrol solutions increases the ionic strength of the medium to an extent that causes dissociation of nuclear ribosomes, breaking the 78-S component into smaller units (see Fig. 5). For this
A CRUDE RIBOSOMES
--
115S 75S65S45S
D RIBOSOMES TREATED WITH DO(3
• Jl
C RIBOSOMES TREATED WITH NoCI
66S 40-55S
Fig. 5- U l t r a c e n t r i f u g a l p a t t e r n s of n u c l e a r r i b o s o m e fractions. N o t e t h e presence of a 75-s c o m p o n e n t , A, w h i c h b r e a k s d o w n w h e n r i b o s o m e s u s p e n s i o n s are t r e a t e d w i t h L u b r o l + N a C 1 , B, o. 5 M NaC1 alone, C, or w i t h d e o x y c h o l a t e a t ionic s t r e n g t h o.I, D. All p a t t e r n s o b t a i n e d a t 37 020 r e v . ] m i n ; A a n d D, a f t e r i o m i n a t 80°; B, a f t e r 18 rain a t 8°; C, a f t e r IO rain a t 25 °. I n p a t t e r n ]3, 64 S a n d 42 S c o r r e s p o n d to 75 S a n d 55 S w h e n m e a s u r e d in m o r e d i l u t e s u s p e n s i o n s . F o r f u r t h e r details see t e x t .
reason further purifications using Lubrol-salt combinations were not attempted, and we proceeded to test the effects of ionic detergents such as sodium deoxycholate, avoiding high salt concentrations. Sodium deoxycholate: The use of this substance in solubilizing the membrane fraction of rat-liver microsomes was first reported by STRITTMATTERAND BALL in their work on microsomal piglnents 31, and deoxycholate has been wide used elsewhere in the purification of the ribonucleoprotein particles of the cytoplasm32, sS. In tests on thymus nuclear ribosomes, it was found that much of the protein associated with the crude ribosomal pellet (Fraction I) can be solubilized b y treatment with sodium deoxycholate in the concentration range o.5-1.o %. Two conditions have been employed, depending on the ionic strength of the Tris buffer used in the initial extraction of the nuclei. Our first experiments were carried out at ionic strengths near o.I. The ribosomes were extracted in o.I M Tris buffers at p H 7.6 containing 5" lO-4 M MgC1v The crude ribosomal pellet (Fraction I) was resuspended in o.ooiI M sodium phosphate butter (pH 7.6) containing 5 . I o - 4 M MgCI~ and o.I/l~r KC1 (to maintain high ionic strength). The ribosome concentration of the susBiochim. Biophys. Acta, 55 (1962) 8 4 9 - 8 6 4
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A.O. POGO, et al.
pensions ranged from IO to 20 mg/ml. The suspensions were dialyzed against the buffer-KC1 mixture for 20 h at 2 °, and large particles were then removed by centrifugation at 15 ooo × g for 15 min. To the supernatant fraction, containing the ribosomes, was added a concentrated solution of sodium deoxycholate in buffer. (It is convenient, in carrying out this operation, to prepare a stock solution of 5 % DOC in water and to mix appropriate volumes of this solution with the buffer immediately before use. This avoids the formation of precipitates which occurs when deoxycholate and Mg~+ containing buffers are mixed and allowed to stand for prolonged periods.) In all our experiments an amount of deoxycholate was added to exceed the weight of ribosomes in the suspension by ten-fold. Nuclear ribosome suspensions became completely clear after standing for 20-30 min at 2 ° in the presence of deoxycholate. They were then centrifuged at lO5 ooo X g for 2 h. The pellet, consisting of purified ribosomes, occurs as a clear, firm gel. A small, overlying white sediment, which is not ribosomal, could be readily removed by washing the pellet with a small amount of buffer, swirling and decanting the white slurry. The clear pellet could then be transferred with the aid of a spatula to a glass homogenizing tube and then resuspended in the buffer using a slowly-rotating Teflon pestle. For purposes of analysis, aliquots of this suspension were added to equal volumes of 20 % trichloroacetic acid, and the precipitates treated with ethanol, ethanol-ether, and ether, as described previously. The composition of nuclear ribosomes treated with DOC in this way is given in Table I. Their high RNA content (62 %) is similar to that described for bacterial ribosomes by TISSIERES AND WATSON25, who analyzed the ribosomes of Escherichia coli and found them to contain 60-65 % RNA. The ribonucleic acid content of nuclear "DOC"-ribosomes is considerably higher than most figures reported for cytoplasmic ribosomes from other animal tissues. Guinea-pig liver ribosomes, for example, contain 40-45 % RNA, even though deoxycholate is used in the isolation ~. The increase in RNA content from 24.5 % in the crude ribosomal pellet (Fraction I) to over 60 % in nuclear "DOC"-ribosomes indicates that detergent treatment has removed much of the protein without concomitant losses in RNA content. Although the protein solubilized by deoxycholate is not all derived from ribosomes, part of it must have been associated with the ribosomes in a functional way. Thus the extract includes many amino acid "activating" enzymes. Moreover, tracer experiments using 14C labeled amino acids show that deoxycholate solubilizes many of the proteins synthesized by ribosomes which are still weakly associated with the synthetic site 5. Our first isolations of nuclear DOC-ribosomes were carried out at ionic strengths near o.I. It was soon found that the method required modification because examination of these preparations in the analytical ultracentrifuge showed the bulk of the material now appeared as a 48-S component. The relatively high ionic strength employed in the isolation had caused a dissociation of the 78-S component into the smaller units (see Fig. 5). To avoid this, subsequent isolations of nuclear ribosomes were carried out at much lower ionic strengths, extracting the ribosomes in o.oi M "iris buffers containg 5" lO-4 M MgCI~, and subsequently treating the crude ribosome pellet with 1 % deoxycholate in o.ooli M sodium phosphate-o.ooo5 M MgC1, (pH 7.6). Nuclear ribosomes prepared in the presence of deoxycholate at these low ionic Biochim. Biophys. dcta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
859
strengths occur largely as a 78-S component (Fig. 6). Their chemical composition is given in Table I, which presents the average of five different preparations under these conditions. Again it can be seen that RNA comprises about 6o % of the dry weight of TCA-washed ribosomes.
A DOG RIBOSOMES
103S ~ S S T S B UNTREATED RIBOSOMES
~PLUS EDTA
C ~lC
RIBOSOMES PLUS EDTA
,
t
Fig. 6. Ultracentrifugal patterns of nuclear ribosomes, showing effects of Mg~+ removal on ribosome structure. Note the presence of the 72-s component in purified ribosomes, A, which disappears when t~DTA is added, C. A similar effect is seen for crude ribosome suspensions {Compare (B) with Fig. 5 A). All patterns obtained at 37 o20 rev./min; A, after 14 min at 9°; B and C, after 18 rain at 8°. For further details see text. As in the case of cytoplasmic and bacterial ribosomes ~3-~s, the structure and sedimentation properties of the purified nuclear ribosomes depend to a large extent on the magnesium ion concentration of the suspending medium. At 5" lO-4 M Mg2+, the particles occur largely as a 78-S component. Removal of magnesium by dialysis against solutions of a chelating agent (e.g. EDTA) leads to an extensive disruption of the 78-s component, with the appearance of the smaller units of about 50 s, 42 s, and 28 s (Fig. 6). When EDTA-treated ribosomes are collected by centrifugation at lO5 ooo × g for 4 h, a pellet is obtained consisting of these smaller subunits. Analysis of the pellet shows the RNA content remains at about 60 % (Table I). Further details on the sedimentation properties of nuclear ribosomes will be presented below.
Nucleotide composition o/ ribosomal ribonucleic acid The average base composition of the RNA in nuclear ribosomes isolated with the aid of sodium deoxycholate has been determined. For this purpose 5-1o mg of ribosomes (dry wt.) were hydrolyzed in I N NaOH for 22 h at 30 °. The nucleotides were
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A.O. POGO, et al.
separated b y paper chromatography in the NH3-isobutyric acid solvent system of ELSON et al) s. The nucleotide composition of the ribosomal RNA is compared with that of the total nuclear RNA in Table II. It can be seen that the overall compositions are quite similar, and that ribosomal RNA has the usual high proportions of guanylic TABLE II NUCLEOTIDE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID FRACTIONS Moles]zoo moles total nucleotide present as Material analyzed
Adenylic acid
Uridylic acid
Total nuclear R N A " N u c l e o l a r " R N A fraction Nuclear ribosomal 1RNA Nuclear " M e s s e n g e r " R N A
20.4 21.8 20. 4 29.3
19.3 23.5 18.6 28.4
33.3 3o.5 32.7 22.5
27.8 25.7 28.2 19.8
Thymidylic 28. 3
22.6
19.9
T h y m u s DNA*
27.6
Guanylie acid
Cytidylic acid
Relerence
(27) (36)
* F o r p u r p o s e s of comparison.
and cytidylic acids. It should be stressed however, that not all nuclear RNAs have the same base ratios, and that thymus nuclear RNA includes a small but metabolically very active fraction, which is low in guanylic and cytidylic acids, and high in adenylic and uridylic acids, and thus resembles thymus DNA in its base composition (if one considers RNA-uridylic acid equivalent to DNA thymidylic acidae). Electron microscopy o[ nuclear ribosomes Ribosomes isolated in the presence of sodium deoxycholate have been examined under the electron microscope. In preparing these ribosomes for electron microscopy, the DOC-ribosome pellet was resuspended in o.ooli M sodium phosphate-o.ooo5 M MgClz at pH 7.6. Droplets of the suspension were transferred directly to "formvar" films on copper grids. The liquid phase was rapidly absorbed by sintered glass discs placed beneath the grids. The ribosomes on the film were further washed free of sucrose by washing three times with o.I-ml portions of the magnesium-containing buffer. After drying, the films were shadow-cast with chromium at an angle of 15 ° . The pictures were taken in the Hitachi Model H U - I I electron microscope at plate magnifications of 20 ooox and 40 ooox. In one experiment, the DOC-ribosomes were further purified by centrifugation in a sucrose density-gradient, as described earlier. After locating the ribosome peak by its absorption at 260 m/~, o. I-ml aliquots of the peak solution were transferred to "formvar" films on copper grids. The ribosomes were washed and shadow-cast with chromium, as described above. The results are shown in Fig. 7, A and t3. It can be seen that nuclear ribosomes are roughly spherical or ellipsoidal and that several size classes are represented in the pictures. When dimensions are measured perpendicular to the direction of shadowing, it is found that most of the particles are about 260/~. units wide, but others range in size from 13o A to 350 A. The 26o-A particles are considered to represent Biochim.
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the 78-s ribosomes, since the latter comprise the most prevalent component in ultracentrifugal analysis. The dimension 260 A includes the cap of chromium metal deposited by shadowcasting; this exaggerates the apparent width of the particle, as was pointed out for E. coli ribosomes by HALL AND SLAYTER37. They found that shadow-casting with
Fig. 7. E l e c t r o n m i c r o g r a p h s of t h y m u s n u c l e a r ribosomes. T h e r i b o s o m e s were p r e p a r e d w i t h d e o x y c h o l a t e a t low ionic s t r e n g t h s a n d t h e n purified in a sucrose d e n s i t y - g r a d i e n t . D r o p l e t s of t h e purified r i b o s o m e s u s p e n s i o n were dried on " F o r m v a r " f i l m s a n d s h a d o w e d w i t h c h r o m i u m a t a n a n g l e of 15 ° . F o r f u r t h e r details see t e x t .
platimum increased the dimensions of E. coli ribosomes by about 60 A. If a similar correction is made in the case of thymus nuclear ribosomes, the corrected width is about 200/~. (This dimension is still considerably higher than the figures previously reported for nuclear ribosomes fixed in situ with osmium tetroxidO; osmium-fixed ribosomes are usually lOO-15o/~ in diameter. The discrepancy is probably due to the great differences in fixation method and preparation of samples.) It is of interest that both thymus nuclear ribosomes (78 s) and E. coli 7o s ribosomes are about 200 A across, and that both contain the same relative proportions of RNA and total protein. Ultracentri/ugal analysis o/ thymus nuclear ribosomes Before analysis, nuclear ribosomes were dialyzed overnight at 2° against buffer systems commonly employed in the study of cytoplasmic ribosomes. Two systems were used (a) o.I M KCl-o.oolI M potassium phosphate, pH 7.4-o.ool 5 M MgCI~, and (b) o.ooli M potassium phosphate, pH 7.4, containing 0.0005 M MgCI~; this Biochim. Biophys. Acfa, 55 (I962) 8 4 9 - 8 6 4
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A.O. POGO, et al.
was made up to o.o3 M in KC1 just before analysis. These systems will be referred to as "o.I" and "0.03" ionic strength buffers. The samples were centrifuged in 12 ram, 2 ° double-sector cells at 37 020 rev./min, and, unless otherwise indicated, analyses were performed at 8 °. The schlieren pictures were enlarged and traced. Sedimentation coefficients were calculated in the usual way and the concentrations were computed from the areas under the curves, assuming a specific refractive index increment of o.oo187 for a I °/o solution. Analysis of the crude ribosomal pellet (Fraction I) shows that it consists of a mixture of particles of differing sedimentation properties. At ionic strengths near o.I the schlieren pattern consistently showed 4 or 5 ribosome peaks as well as a large, slowly-sedimenting boundary (Fig. 5 A). The chief component (which accounted for approx. 4 ° ~o of the total pattern area) had a sedimentation constant of about 75 S (uncorrected) at a total concentration of 4 mg/ml. The other components had sedimentation constants of 115 S, 65 S, and 45 S. This pattern is similar to that obtained for ribosomes from a variety of other sources (see ref. 38). In addition to the ribosome peaks, smaller components with sedimenta[ion constants near IO s, and occasionally a 2o-S component were observed; these accounted for about 30 % of the total area of the pattern. (A 2o-S component has also been found b y PETERMANN in unpurified Jensen sarcoma ribosomes but was shown to be lacking in ribonucleic acid~9).
E][ects o/detergents and salts on nuclear ribosome structure Lubrol-NaCl: Examination of ribosome suspensions prepared with the aid of Lubrol-salt solutions showed evidence of extensive dissociation of the particles. Although essentially the same components were observed as in untreated ribosomes, their relative proportions had changed considerably (Fig. 5 B). The 75-S ribosomes were present in much lower concentration than in untreated samples; instead a 55-S peak began to predominate in the schheren pattern. The II5-S component was not a distinct peak but a mixture of polydisperse materials. Also present was IO S material, in approximately the same percentage as in untreated ribosome suspensions. Attempts to reverse the dissociation by doubling the magnesium ion concentration (see below) had only a small effect, augmenting the 75-S component and decreasing the 55-S component shghtly. (Similarly, the addition of spermidine phosphate to a final concentration of 0.005 M removed the 55-S peak and led to the appearance of 69-7I-S components and some polydisperse heavy material.) Since the Lubrol treatment is carried out in the presence of 0.5 M NaCI, the effect of salt alone was tested, with the results shown in Fig. 5 C. The pattern shows almost complete conversion of the 75-S ribosomes to material with sedimentation values between 33 S and 40 S, and a sizeable increase in the percentage of Io-S material This it seems clear that the Lubrol+salt treatment is damaging. Sodium deoxycholate: A similar examination of the effects of deoxycholate on the nuclear ribosomes revealed a marked dependence on the ionic strength of the medium. Ribosomes prepared with DOC at ionic strengths near o.I gave the pattern shown in Fig. 5 D. Although a 74-S component was present, the bulk of the pattern was made up of a 48-S component. (As in the case of Lubrol-NaC1 treated ribosomes,'the addition of spermidine phosphate to these DOC ribosomes seemed to convert the 48-S Biochim. Biophys. Acta, 55 (1962) 849-864
PURIFICATION OF NUCLEAR RIBOSOMES
863
material to larger units, thus increasing the 74-S peak and the fast polydisperse boundary. ) When the ribosomes were extracted from the nuclei in very dilute Tris buffers (o.oi M) and subsequently treated with deoxycholate at low ionic strengths, much less dissociation was observed. Under these conditions of low ionic strength, 7° ~ of the pattern consisted of a 72-S component; in addition there was 15 % of a Io3-S component and 15 % of 57-S material (Fig. 6 A). No slowly sedimenting material was observed. The sedimentation coeHicient o[ nuclear DOC-ribosomes Ribosomes prepared with the aid of deoxycholate at low ionic strengths were analyzed in the ultracentrifuge at three concentrations, 3.1, 1.2, and 0. 9 mg/ml (keeping the ionic strength constant at 0.03). Schlieren optics were used. The sedimantation coefficients, s20,w, for the three analyses were 71.8, 74.2 and 74.6. These extrapolated to a value of s20,w, of 75.6- Thus nuclear ribosomes resemble other ribosomes in the strong concentration dependence of their sedimentation rates. The DOC-ribosomes were also examined with ultraviolet optics at a concentration close to o.05 mg/ml. The sedimentation coefficient in this case was 77-9 S. Thus, with a sedimentation coefficient at infinite dilution of about 78 S, nuclear ribosomes fall into the rather large class of 8o-S ribosomes. Role o[ Mg 2+ ions in nuclear ribosome structure All ribosomes seem to be composed of nucleoprotein subunits held together by magnesium ions. The role of Mg2+ in nuclear ribosome structure was made evident in experiments in which the chelating agent, EDTA, was added. In untreated ribosome suspensions, in which the 74-S component predominates, small amounts of EDTA led to the appearance of 64-S and 42-S components; at o.oi M EDTA only 49-S and 33-S components were present (Fig. 6 B). In the deoxycholate-treated ribosomes prepared at low ionic strengths, EDTA acted in a similar fashion, disrupting the 72-74 S component completely to units of 42 S and 28 S (compare Figs. 6 C and 6 D). These results resemble those obtained by PETERMANN AND HAMILTONwith ratliver ribosomes88, where the 83-S component breaks down into particles of 46 S and 30 S when the Mg2+ concentration is lowered. Thus, the 78-S nuclear ribosome, like many other ribosomes, appears to be an aggregate of smaller subunits, and magnesium ions play a role in this aggregation.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. M. L. PETERMANN of the Sloan-Kettering Institute for her advice and for many helpful discussions. This research was supported in part by a Grant (RG 4919) from the U.S. Pubhc Health Service. The first Author is a Fellow of the Guggenheim Foundation, and B. G. T. P. a Fellow of the Consejo Nacional de Investigaciones Cientlficas y T6cnicas, of the Argentine Republic. V. C. L. and M. G. H. were supported by the United States Atomic Energy Commission, the latter under their Contract AT(3o-I)-9io. Biochim. Biophys. Acta, 55 (I962) 849-864
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A.O. POGO, et al. REFERENCES
' J. W. HOPKINS, Proc, Natl. Acad. Sci. U.S., 45 (1959) 1461. 2 V. G. ALLEREY, J. W. HOPKINS, J. H. FRENSTER AND A. E. MIRSKY, Ann. N . Y . Acad. Sci., 88 (196o) 722. a j . W . HOPKINS, V. G. ALLFREY AND A. E. MIRSKY, Bioehim. Biophys. Acta, 47 (1961) 195. 4 j. H. FRENSTER, V. G. ALLFREY AND A. E. 1VilRSKY, Proc. Natl. Acad. Sei. U.S., 46 (196o) 432. 5 V. G. ALLFREY, A. O. POGO, B. G. T. PoGo AND A. E. ~V[IRSKY, m a n u s c r i p t in p r e p a r a t i o n . 6 K. R. PORTER, J. Histochem. Cytochem., 2 (1954) 346. 7 W. BERNHARD, A. BAUER, A. GROPP, F. HAGENAU AND C. OBERLING, Exptl. Cell Kesearch, 9 (1955) 88. s j . G. LAFONTAINE, J. Biophys. Biochem. Cytol., 4 (1958) 229. 9 j . G. GALL, J. Biophys. Biochem. Cytol. Suppl. 2 (1956) 4o7 . 10 S. WlSCHNI~ZER, J. Ultrastruct. Research, I (1958) 2Ol. n M. L. WATSON, J. Biophys. Biochem. Cytol., 6 (1959) 147. 1, W. BEERMAN AND G. F. BAHR, Exptl. Cell Research, 6 (1954) 195. 13 H. G. CALLAN, Symposium on Fine Structure o/ Cells (Leiden), I n t e r n . Union Biol. Sci. Publ. B2I (1956 ) 89. 14 V. G. ALLFREY, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1957) 451. 15 T. Y. WANG, Biochim. Biophys. Acta, 45 (196o) 8. x8 V. G. ALLFREY, R. MEUDT, J. W. HOPKINS AND A. E, MIRSK¥, Proc. Natl. Acad. Sei. U.S., 47 (1961) 9o7 . x7 H. SACHS, J. Biol. Chem., 228 (1957) 23. 18 V. G. ALLFREY, M. M. DALY AND A. E. MIRSKY, J. Gen. Physiol., 37 (1953) 157. IS Z. DISCHE AND E. BORENFREUND, Biochim. Biophys. Acta, 23 (1957) 639. s0 K. BURTON, Biochem. J., 62 (1956) 315 • 21 Z, DlSCHE, Mikrochemie, 8 (193 o) 4. 22 C. F. CRAMPTON, R. LIPSHITZ AND E. CHARGAFF,J. Biol. Chem., 206 (1954) 499. ,3 p, O. P. T ' s o , J. BONNER AND J. VINOGRAD, Biochim. Biophys. ~'tcta, 3 ° (1958) 57 o. 38 M. L. PETERMANN, J. Biol. Chem., 235 (196o) 1998. ,s A. TlSSI~RES AND J. D. WATSON, Nature, 182 (1958) 778. 26 l-I. ~AORA, H. NAORA, A. E. MIRSKY AND V. G. ALLFREY, J. Gen. Physiol., 44 (1961) 713 • ,7 V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Aead. Sei. U.S., 43 (1957) 821. is R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. 38 p, COHN AND J. A. V. BUTLER, Biochem. J., 7 ° (1958) 254. 30 R. RENDI AND T. HULTIN, Exptl. Cell Research, 18 (1959) 542. 31 C. F. STRITTMATTER AND E. G. BALL, Proc. Natl. Acad. Soc. U.S., 38 (1952) 19. 32 j . W. LITTLEFIELD, E. B. KELLER, J. GRoss AND P. C. ZAMECNIK, J. Biol. Chem., 217 (1955) 11. 3a p. O. P. T s ' o AND J. VINOGRAD, Biochim. Biophys. Acta, 49 (1961) 113. 34 j . F. KIRSCH, P. SIEKEVlTZ AND G. E. PALADE, J. Biol. Chem., 235 (196o) 1419. a3 D. ELSON, T. GUSTAFSON AND E. CHARGAEF, J. Biol. Chem., 209 (1954) 285. 3s A. SIBATANI, A. E. ~/[IRSKYAND V. G. ALLFREY, Proc. Natl. Acad. Sci. U.S., in t h e press. 37 C. E. HALL AND H. S. SLAYTER, J. Mol. Biol., i (1959) 329. 3s ~VI.L. PETERMANN AND M. G. HAMILTON, in R. J. C. HARRIS, Physicochemical Studies on Ribonueleoproteins [rom Mammalian Cytoplasm, in Protein Biosynthesis, L o n d o n Academic Press, Ltd., 1961, p. 233. 3, IV[. L. PETERMANN, personal c o m m u n i c a t i o n .
Biochim. Biophys. Acta, 55 (1962) 849-864