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Reversible dissociation of nuclear ribonucleoprotein particles containing mRNA into RNA and protein
J. HoZ. Biol. (1967) 2’7, 187-191 LETTERSTOTHEEDITOR
Reversible Dissociation of Nuclear Ribonucleoprotein Particles containing mRNA into RNA and Protein It has been shown that the major part of mRNA in cell nuclei is present in special ribonucleoprotein particles. These partioles are homogeneous, have a sedimentation coeiIicient of about 30 s, and contain mRNA and protein in the ratio of 1: 4 to 1: 5. They are of discoidal shape with dimensions of about 18OA x 18OA x SOA. They can combine with some additional amounts of free mRNA, this reaction being specific for mRNA (but not for ribosomal RNA). It has been suggested that these particles participate in the transport of mRNA from chromosomes to cytoplasm (Samarina, Asrijan & Georgiev, 1965; Samarina, Krichevskaya & Georgiev, 1966, 1967b; Samarina, Krichevskaya, Molnar, Bruskov & Georgiev, 1967a.) To obtain further information on the structural organization of these particles, attempts were made to dissociate their mRNA and protein. In the work to be reported here, this aim has been achieved by treatment of the 30 s particles with salt solutions of high ionic strength, or with urea. Removal of the dissociating agents leads to reconstitution of about half the particles by self-assembly. Nuclear mRNA-protein particles were isolated from rat liver nuclei as previously described (Samarina et al., 19651967a). For this purpose the nuclei, after purification in concentrated sucrose solution, were extracted with O-14 ?d-NaCl-O*OOl M-MgC!l,0.01 M Tris (STM) at pH 7 and then at pH 8. The second and third extracts obtained at pH 8 containing the major part of the nuclear mRNA were combined and ultracentrifuged through a 15 to 30% sucrose gradient in 0.1 M-NaC1-0901 M-MgC1,-0905 ~-sodium phosphate (pH 7.0) (SPM). Sedimentation coefficients of peaks were determined according to Martin & Ames (1961) using the ribosomal RNA (28 s and 18 s peaks) or ribosomal subunits (30 s and 50 s), as standards. The 30 s peak was collected from the gradient (Fig. l(a)). One portion of the material was fixed by addition of 10% formaldehyde to a final concentration of 2%. The fixed material was recentrifuged in a sucrose gradient (15 to 30% sucrose in SPM, containing 1% formaldehyde and in a preformed CsCl density gradient (Spirin, Belitsina & Lerman, 1965) (O-005 M-sodium phosphate (pH 8.0) with 2% formaldehyde). The results of these recentrifugations are shown in Fig. l(b) and (c). It can be seen in Fig. l(b) that the fixed particles have the same sedimentation properties as unfixed particles (30 s). Figure l(c) shows that the buoyant density of the fixed particles is about l-40 (between 1.385 and l-410 in different experiments). Two further portions of the material collected as the 30 s peak of the first sucrose density-gradient (Fig. l(a)) were made O-7 M in KC1 or 25 M in NaCl. One-half of each of these portions was dialysed against SPM and then fixed by 2% formaldehyde. The other halves were first fixed with 2% formaldehyde and then dialysed against SPM-2% formaldehyde. The preparations obtained in this way were then ultracentrifuged in a sucrose gradient, or in a CsCl density gradient, under the same conditions as untreated particles. The results presented in Fig. l(d), (e) and (f) show that treatment with either IS7
2
-O
0-
0-
7 0
8
LETTERS
TO THE
EDITOR
189
O-7 ~-Kc1 or 2.6 ~-Nacl causes the disappearance of the 30 s particles, their protein (determined in these experiments on the basis of o,~.~eo) and RNA being recovered in the light (4 to 6 s) zone of the suorose gradient or randomly distributed in the CsCl gradient. The results presented in Fig. l(g) and (h) show that dialysis prior to fixation causes reunion of about 60% of mRNA and protein and reconstitution of 30 s particles. As can be seen in Fig. l(i) and (l), the buoyant density of the reoonstituted particles is equal to that of the original particles (p = 1.40). Eleotron miorographs of the original 30 s particles and the material of the peak passed through 2.5 M-N~CI and subsequently dialysed against SPM (before formaldehyde fixation) are presented in Plate I. They show that many of the particles obtained after the dissociation-reconstitution procedure are similar to the original 30 s particles in respect to their shape and dimensions. After purhlcation by ultracentrifugation in a sucrose gradient, these reconstituted particles are concentrated in the 30 s band and can be seen to be a much more homogeneous material (Plate I(c)). Very similar dissociation-reassociation reactions were observed upon addition of urea to 30 s part&lea to 4 M final concentration and subsequent removal of urea by dialysis (Fig. l(j) and (k)). In this case formaldehyde was not added, and in the experiment recorded on Fig. l(j) the sucrose gradient contained 4 ~-urea. These experiments do not, however, prove the dissociation of RNA and protein of the 30 s particles, since the similarity in sedimentation coefficients of these two components after salt and urea treatment prevents their separation. To settle this point, the material of the 30 s peak (isolated from rat liver labelled with 3aP for FIG. 1. Sedimentation profiles of original, dissociated and reconstituted nuclear particles, containing mRNA. (a) Nuclear extract containing mRNA (rata were injected with [‘*C]orotic acid 30 min before killing. The sedimentation was carried out in 16 to 30% sucrose gradient in SPM (pH 7.2) in a Spinco L SW26 rotor at 23,000 rev./mm, 4”C, for 14 hr). (b) Reeedimentation of the 30 a peak after ita isolation and fixation by formaldehyde (16 to 30% sucrose gradient in SPM-1% formaldehyde, SW39, 36,000 rev./m@ 4’C, 4 hr). (o) Recentrifugation of 30 8 peak in a preformed C&l gradient (p = I.20 to 160) (SW39, 36,000, rev./min, 4’C, 18 hr). (d) Sucrose gradient sedimentation of nuclear particles dissociated by 0.7 M-KC& fixed with 2% formaldehyde and then dialyeed against SPM-1% formaldehyde (SW26, 23,060 rev./m& 16 hr). (e) Sucrose gradient sedimentation of nuclear partioles dissociated by 2.6 w-N&Cl (and then treated aa (d) (SW39,36,000 rev./min, 4 hr). (f) Sedimentation of nuclear particles dissociated by 0.7 ~-Kc1 (and then treated as in (d)) in a preformed C&l gradient (p = 1.2 to 1.6) (SW39. 36,000 rev./mm, 18 br). (g) Suoroae gradient sedimentation of particles reconstituted by dialyaia against SPM after their prior dissociation in 0.7 ar-KCl. After dialysis the particles were fixed with 2% formaldehyde (SW26, 23,000 rev./min, 14 hr). (h) The same as (g) but reconstitution after prior dissociation in 2.6 M-N&~ (SW39, 36,000 rev./mill, 4 hr). (i) Ultracentrifugation of the material shown in (g) in a CsCl density-gradient (p = 1.20 to I-60, SW39, 36,000 rev./min, 18 hr). (j) Dissooiation of 30 8 peak in 4 y-urea. The 16 to 30% sucrose gradient contains 4 ~-urea and O-006 M-K&PO* (pH 7.2) (SW39, 36,000 rev./min, 4 hr). (k) Reaarociation of the 30 8 peak by dialysis in SPM after its dissociation in 4 M-urea (16 to 30% suarose gradient on SPM, SW39, 36,000 rev./m& 4 hr). (1) Ultracentrifugation of the forma&&r&material aa in (h) in a C&l density-gradient (p = 1.26 to 1.66, SW39, 36,000 rev./min, 16 hr). -a-@--, Ultraviolet absorption at 280 mp; --A--A--, ultraviolet absorption at 230 rnp; -O-O-, radioaotivity of a&d-insoluble fraotion. The arrows indicate the position at which the 30 8 peak should be Fecovered (determined on the b&s of the parallel sedimentation of markers : ribosomea or ribosomal RNA).
190
0. P. SAMARINA
ET
AL.
18 hours and with [‘*C]orotic acid for 30 minutes) was mixed with carrier high molecular weight ribosomal RNA (2 mg RNA per ml.) and NaCl was added to 2-6 M concentration. The mixture was then dialysed against 2.5 M-NaCl to remove sucrose and the RNA precipitated was removed by centrifugation at 6000 g for 1.5minutes and washed by 2-5 M-NaCl. The distribution of total RNA (3aP), rapidly labelled RNA (14C) and total protein (Lowry, Rosebrough, Parr & Randall, 1951) between precipitate and supernatant fraction was analysed (Table 1). About 95% of the 14C was recovered in the precipitate and 90 to 93% of protein in the supernatant fraction. TABLE
Separation of RNA and protein No. of exp.
1
2
Material
Origin81 30 8 particles Dissociated particles in 2.6 na-NaCl o/0 dissociation Origin81 30 9 particles Dissociated psrticles in 2.5 M-NcCl o/0 dissociation
1
from 30 s particles
in 2.5 x-NaCl
RNA radioactivity 1.T cts/min saP
assayed
393
Precipitate -l Supernatant
374 9 97.5 608 560 31 95.0
Precipit8te { Supernat8nt
0.4
100
0.2
2 T 3 .L! x .e .; ” B xi 100 2
s: s5 d6
0.4
1060 894 151 86.0 3160 2740 279 86.0
Protein (PEd 576 36 520 93.0 140 13 124 90-9
0.2
IO Fraction
IS
no.
FIG. 2. Reconstitution of nuclear particles with exogenous RNA. The 30 s pesk was dissoci8ted by 2.6 M-N&~ in the presence of unlabelled ribosomal RNA as carrier (2 mg/ml.). In these conditions 8n RNA precipitate W&S formed. It w&s removed by centrifug8tion. and labelled 18 s mRNA w8s added to the supernat8nt fraction (N 75 pg). One part (8) was fixed with formaldehyde and dialysed against SPM-1% formaldehyde and another (b) dialysed and then Exxed. Ultraeentrifugrttion was performed throIlgh 8 16 to 30% sucrose gr8dient in the SW39 rotor at 36,000 rev./m& 4 hr. The 18 s mRNA w&8 obt8ined by sucrose gr8dient sedimentation of SaP&belled mRNA of rat liver isolated by the hot phenol fractionation method (Georgiev, Sam8rin8, Lerm8n & Smirnov, 1963).
PLATE I. Electron micrographs of (a) original particles; (b) material reconstituted from 2.5 ~-N&cl by dialysis against SPM; and (c) the purified 30 s peak, obtained from (b) by sucrose gradient ultracentrifugation. All preparations after fixation with 2% formaldehyde were dialysed against 1% formaldehyde-O.005 M-phosphate (pH 7.0). The specimens were stained negatively with uranyl acetate (a), (c), or with phosphotungstic acid (b). Total magnification 200,000. [“facing
p. 190
LETTERS
TO THE
EDITOR
191
The recovery of saP in the precipitate is about 85%, the lowerrecovery compared to 14C probably representing the incorporation of some radioactive phosphate into proteins. Thus physical separation of particulate RNA and protein was achieved. An excess of free mRNA ( - 18 s) labelled with 14Cwas then added to the supernatant fraction. The mixture was divided in two parts. One part was dialysed against SPM and fixed with formaldehyde (Fig. 2(b)), and the other part 6rst fixed with formaldehyde and then dialyzed (Fig. 2(a)). The sedimentation proties obtained after both treatments indicate that if dialysis precedes fixation, the free mRNA added is incorporated to a limited extent into reconstituted particles. This low yield may be due to the interference of non-labelled carrier during reconstitution. Essentially the same results were obtained in experiments in which the labelled mRNA isolated from the 30 s peak was added to the mixture, instead of 18 s mRNA (prepared by a hot phenol extraction method). These experiments demonstrate that the 30 s particles may be dissociated into their protein and RNA components and then reconstituted by self-assembly after removal of dissociating agent. Dissociation breaks the particles into the relatively small protein subunits (with sedimentation coe&cients of 4 to 6 s). A full account of this work will be published in Molecular Biology (U.S.S.R.). We thank Dr A. S. Spirin, in whose laboratory the first experiments on C&l ultrscentrifugation were performed, for his encouragement and 0. V. Ago1 for valuable technical assistance. Institute of Molecular Biology Academy of Sciences of the U.S.S.R. Moscow, U.S.S.R.
Received 29 December
0.
P. SAMARINA
J. E. V. A. G.
MonNaRt M. bKANIDIN I. BRUSKOV$ A. KRICEEVSKAYA P. GEORCIEV
1966, and in revised form 27 March 1967 REFERENCES
Georgiev, G. P., Samarina, 0. P., Lerman, M. I. & Smirnov, M. V. (1963). Nature, 200, 1291. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. 8z Randall, R. J. (1951). J. Biol. Chem. 193, 265. Martin, R. G. & Ames, B. N. (1961). J. Riol. Chem. 226, 1372. Samarina, 0. P., Asrijan, I. S. & Georgiev, G. P. (1965). Dokl. Alcud. Nauk U.S.S.R., 168, 1510. Sama1511a, 0. P., Krichevskaya, A. A. & Georgiev, G. P. (1966). Nature, 210, 1319. Samarina, 0. P., Krichevskaya, A. A., Molnar, J., Bruskov, V. I. t Georgiev, G. P. (1967a). Mol. Bid. U.S.S.R., 1, 129. Samarina, 0. P., Krichevskaya, A. A. & Georgiev, G. P. (19676). Mol. Biol. U.S.S.R., I, N7 in the press. Spirin, A. S., Belitaina, N. V. & Lerman, M. I. (1966). J. Mol. Biol. 14, 611.
t Present address: Medical University of Pets, Hungary. $ Institute of Crystallography, Academy of Sciences of the U.S.S.R., Moscow.
Reversible Dissociation of Nuclear Ribonucleoprotein Particles containing mRNA into RNA and Protein It has been shown that the major part of mRNA in cell nuclei is present in special ribonucleoprotein particles. These partioles are homogeneous, have a sedimentation coeiIicient of about 30 s, and contain mRNA and protein in the ratio of 1: 4 to 1: 5. They are of discoidal shape with dimensions of about 18OA x 18OA x SOA. They can combine with some additional amounts of free mRNA, this reaction being specific for mRNA (but not for ribosomal RNA). It has been suggested that these particles participate in the transport of mRNA from chromosomes to cytoplasm (Samarina, Asrijan & Georgiev, 1965; Samarina, Krichevskaya & Georgiev, 1966, 1967b; Samarina, Krichevskaya, Molnar, Bruskov & Georgiev, 1967a.) To obtain further information on the structural organization of these particles, attempts were made to dissociate their mRNA and protein. In the work to be reported here, this aim has been achieved by treatment of the 30 s particles with salt solutions of high ionic strength, or with urea. Removal of the dissociating agents leads to reconstitution of about half the particles by self-assembly. Nuclear mRNA-protein particles were isolated from rat liver nuclei as previously described (Samarina et al., 19651967a). For this purpose the nuclei, after purification in concentrated sucrose solution, were extracted with O-14 ?d-NaCl-O*OOl M-MgC!l,0.01 M Tris (STM) at pH 7 and then at pH 8. The second and third extracts obtained at pH 8 containing the major part of the nuclear mRNA were combined and ultracentrifuged through a 15 to 30% sucrose gradient in 0.1 M-NaC1-0901 M-MgC1,-0905 ~-sodium phosphate (pH 7.0) (SPM). Sedimentation coefficients of peaks were determined according to Martin & Ames (1961) using the ribosomal RNA (28 s and 18 s peaks) or ribosomal subunits (30 s and 50 s), as standards. The 30 s peak was collected from the gradient (Fig. l(a)). One portion of the material was fixed by addition of 10% formaldehyde to a final concentration of 2%. The fixed material was recentrifuged in a sucrose gradient (15 to 30% sucrose in SPM, containing 1% formaldehyde and in a preformed CsCl density gradient (Spirin, Belitsina & Lerman, 1965) (O-005 M-sodium phosphate (pH 8.0) with 2% formaldehyde). The results of these recentrifugations are shown in Fig. l(b) and (c). It can be seen in Fig. l(b) that the fixed particles have the same sedimentation properties as unfixed particles (30 s). Figure l(c) shows that the buoyant density of the fixed particles is about l-40 (between 1.385 and l-410 in different experiments). Two further portions of the material collected as the 30 s peak of the first sucrose density-gradient (Fig. l(a)) were made O-7 M in KC1 or 25 M in NaCl. One-half of each of these portions was dialysed against SPM and then fixed by 2% formaldehyde. The other halves were first fixed with 2% formaldehyde and then dialysed against SPM-2% formaldehyde. The preparations obtained in this way were then ultracentrifuged in a sucrose gradient, or in a CsCl density gradient, under the same conditions as untreated particles. The results presented in Fig. l(d), (e) and (f) show that treatment with either IS7
2
-O
0-
0-
7 0
8
LETTERS
TO THE
EDITOR
189
O-7 ~-Kc1 or 2.6 ~-Nacl causes the disappearance of the 30 s particles, their protein (determined in these experiments on the basis of o,~.~eo) and RNA being recovered in the light (4 to 6 s) zone of the suorose gradient or randomly distributed in the CsCl gradient. The results presented in Fig. l(g) and (h) show that dialysis prior to fixation causes reunion of about 60% of mRNA and protein and reconstitution of 30 s particles. As can be seen in Fig. l(i) and (l), the buoyant density of the reoonstituted particles is equal to that of the original particles (p = 1.40). Eleotron miorographs of the original 30 s particles and the material of the peak passed through 2.5 M-N~CI and subsequently dialysed against SPM (before formaldehyde fixation) are presented in Plate I. They show that many of the particles obtained after the dissociation-reconstitution procedure are similar to the original 30 s particles in respect to their shape and dimensions. After purhlcation by ultracentrifugation in a sucrose gradient, these reconstituted particles are concentrated in the 30 s band and can be seen to be a much more homogeneous material (Plate I(c)). Very similar dissociation-reassociation reactions were observed upon addition of urea to 30 s part&lea to 4 M final concentration and subsequent removal of urea by dialysis (Fig. l(j) and (k)). In this case formaldehyde was not added, and in the experiment recorded on Fig. l(j) the sucrose gradient contained 4 ~-urea. These experiments do not, however, prove the dissociation of RNA and protein of the 30 s particles, since the similarity in sedimentation coefficients of these two components after salt and urea treatment prevents their separation. To settle this point, the material of the 30 s peak (isolated from rat liver labelled with 3aP for FIG. 1. Sedimentation profiles of original, dissociated and reconstituted nuclear particles, containing mRNA. (a) Nuclear extract containing mRNA (rata were injected with [‘*C]orotic acid 30 min before killing. The sedimentation was carried out in 16 to 30% sucrose gradient in SPM (pH 7.2) in a Spinco L SW26 rotor at 23,000 rev./mm, 4”C, for 14 hr). (b) Reeedimentation of the 30 a peak after ita isolation and fixation by formaldehyde (16 to 30% sucrose gradient in SPM-1% formaldehyde, SW39, 36,000 rev./m@ 4’C, 4 hr). (o) Recentrifugation of 30 8 peak in a preformed C&l gradient (p = I.20 to 160) (SW39, 36,000, rev./min, 4’C, 18 hr). (d) Sucrose gradient sedimentation of nuclear particles dissociated by 0.7 M-KC& fixed with 2% formaldehyde and then dialyeed against SPM-1% formaldehyde (SW26, 23,060 rev./m& 16 hr). (e) Sucrose gradient sedimentation of nuclear partioles dissociated by 2.6 w-N&Cl (and then treated aa (d) (SW39,36,000 rev./min, 4 hr). (f) Sedimentation of nuclear particles dissociated by 0.7 ~-Kc1 (and then treated as in (d)) in a preformed C&l gradient (p = 1.2 to 1.6) (SW39. 36,000 rev./mm, 18 br). (g) Suoroae gradient sedimentation of particles reconstituted by dialyaia against SPM after their prior dissociation in 0.7 ar-KCl. After dialysis the particles were fixed with 2% formaldehyde (SW26, 23,000 rev./min, 14 hr). (h) The same as (g) but reconstitution after prior dissociation in 2.6 M-N&~ (SW39, 36,000 rev./mill, 4 hr). (i) Ultracentrifugation of the material shown in (g) in a CsCl density-gradient (p = 1.20 to I-60, SW39, 36,000 rev./min, 18 hr). (j) Dissooiation of 30 8 peak in 4 y-urea. The 16 to 30% sucrose gradient contains 4 ~-urea and O-006 M-K&PO* (pH 7.2) (SW39, 36,000 rev./min, 4 hr). (k) Reaarociation of the 30 8 peak by dialysis in SPM after its dissociation in 4 M-urea (16 to 30% suarose gradient on SPM, SW39, 36,000 rev./m& 4 hr). (1) Ultracentrifugation of the forma&&r&material aa in (h) in a C&l density-gradient (p = 1.26 to 1.66, SW39, 36,000 rev./min, 16 hr). -a-@--, Ultraviolet absorption at 280 mp; --A--A--, ultraviolet absorption at 230 rnp; -O-O-, radioaotivity of a&d-insoluble fraotion. The arrows indicate the position at which the 30 8 peak should be Fecovered (determined on the b&s of the parallel sedimentation of markers : ribosomea or ribosomal RNA).
190
0. P. SAMARINA
ET
AL.
18 hours and with [‘*C]orotic acid for 30 minutes) was mixed with carrier high molecular weight ribosomal RNA (2 mg RNA per ml.) and NaCl was added to 2-6 M concentration. The mixture was then dialysed against 2.5 M-NaCl to remove sucrose and the RNA precipitated was removed by centrifugation at 6000 g for 1.5minutes and washed by 2-5 M-NaCl. The distribution of total RNA (3aP), rapidly labelled RNA (14C) and total protein (Lowry, Rosebrough, Parr & Randall, 1951) between precipitate and supernatant fraction was analysed (Table 1). About 95% of the 14C was recovered in the precipitate and 90 to 93% of protein in the supernatant fraction. TABLE
Separation of RNA and protein No. of exp.
1
2
Material
Origin81 30 8 particles Dissociated particles in 2.6 na-NaCl o/0 dissociation Origin81 30 9 particles Dissociated psrticles in 2.5 M-NcCl o/0 dissociation
1
from 30 s particles
in 2.5 x-NaCl
RNA radioactivity 1.T cts/min saP
assayed
393
Precipitate -l Supernatant
374 9 97.5 608 560 31 95.0
Precipit8te { Supernat8nt
0.4
100
0.2
2 T 3 .L! x .e .; ” B xi 100 2
s: s5 d6
0.4
1060 894 151 86.0 3160 2740 279 86.0
Protein (PEd 576 36 520 93.0 140 13 124 90-9
0.2
IO Fraction
IS
no.
FIG. 2. Reconstitution of nuclear particles with exogenous RNA. The 30 s pesk was dissoci8ted by 2.6 M-N&~ in the presence of unlabelled ribosomal RNA as carrier (2 mg/ml.). In these conditions 8n RNA precipitate W&S formed. It w&s removed by centrifug8tion. and labelled 18 s mRNA w8s added to the supernat8nt fraction (N 75 pg). One part (8) was fixed with formaldehyde and dialysed against SPM-1% formaldehyde and another (b) dialysed and then Exxed. Ultraeentrifugrttion was performed throIlgh 8 16 to 30% sucrose gr8dient in the SW39 rotor at 36,000 rev./m& 4 hr. The 18 s mRNA w&8 obt8ined by sucrose gr8dient sedimentation of SaP&belled mRNA of rat liver isolated by the hot phenol fractionation method (Georgiev, Sam8rin8, Lerm8n & Smirnov, 1963).
PLATE I. Electron micrographs of (a) original particles; (b) material reconstituted from 2.5 ~-N&cl by dialysis against SPM; and (c) the purified 30 s peak, obtained from (b) by sucrose gradient ultracentrifugation. All preparations after fixation with 2% formaldehyde were dialysed against 1% formaldehyde-O.005 M-phosphate (pH 7.0). The specimens were stained negatively with uranyl acetate (a), (c), or with phosphotungstic acid (b). Total magnification 200,000. [“facing
p. 190
LETTERS
TO THE
EDITOR
191
The recovery of saP in the precipitate is about 85%, the lowerrecovery compared to 14C probably representing the incorporation of some radioactive phosphate into proteins. Thus physical separation of particulate RNA and protein was achieved. An excess of free mRNA ( - 18 s) labelled with 14Cwas then added to the supernatant fraction. The mixture was divided in two parts. One part was dialysed against SPM and fixed with formaldehyde (Fig. 2(b)), and the other part 6rst fixed with formaldehyde and then dialyzed (Fig. 2(a)). The sedimentation proties obtained after both treatments indicate that if dialysis precedes fixation, the free mRNA added is incorporated to a limited extent into reconstituted particles. This low yield may be due to the interference of non-labelled carrier during reconstitution. Essentially the same results were obtained in experiments in which the labelled mRNA isolated from the 30 s peak was added to the mixture, instead of 18 s mRNA (prepared by a hot phenol extraction method). These experiments demonstrate that the 30 s particles may be dissociated into their protein and RNA components and then reconstituted by self-assembly after removal of dissociating agent. Dissociation breaks the particles into the relatively small protein subunits (with sedimentation coe&cients of 4 to 6 s). A full account of this work will be published in Molecular Biology (U.S.S.R.). We thank Dr A. S. Spirin, in whose laboratory the first experiments on C&l ultrscentrifugation were performed, for his encouragement and 0. V. Ago1 for valuable technical assistance. Institute of Molecular Biology Academy of Sciences of the U.S.S.R. Moscow, U.S.S.R.
Received 29 December
0.
P. SAMARINA
J. E. V. A. G.
MonNaRt M. bKANIDIN I. BRUSKOV$ A. KRICEEVSKAYA P. GEORCIEV
1966, and in revised form 27 March 1967 REFERENCES
Georgiev, G. P., Samarina, 0. P., Lerman, M. I. & Smirnov, M. V. (1963). Nature, 200, 1291. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. 8z Randall, R. J. (1951). J. Biol. Chem. 193, 265. Martin, R. G. & Ames, B. N. (1961). J. Riol. Chem. 226, 1372. Samarina, 0. P., Asrijan, I. S. & Georgiev, G. P. (1965). Dokl. Alcud. Nauk U.S.S.R., 168, 1510. Sama1511a, 0. P., Krichevskaya, A. A. & Georgiev, G. P. (1966). Nature, 210, 1319. Samarina, 0. P., Krichevskaya, A. A., Molnar, J., Bruskov, V. I. t Georgiev, G. P. (1967a). Mol. Bid. U.S.S.R., 1, 129. Samarina, 0. P., Krichevskaya, A. A. & Georgiev, G. P. (19676). Mol. Biol. U.S.S.R., I, N7 in the press. Spirin, A. S., Belitaina, N. V. & Lerman, M. I. (1966). J. Mol. Biol. 14, 611.
t Present address: Medical University of Pets, Hungary. $ Institute of Crystallography, Academy of Sciences of the U.S.S.R., Moscow.