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Isolation of nuclear rapidly sedimenting ribonucleic acids by agarose gel filtration
ANALYTICAL
BIOCHEMISTRY
Isolation
of
Nuclear Acids
MILKA Department
367-372 (1972)
49,
Rapidly
by Agarose
B. NOVAKOVIC of Biochemistry,
Sedimenting Gel AND
BOGS Kidlich Beograd, Yugoslavia
Ribonucleic
Filtration S. L. PETROVIC Institute,
P. 0. Box
522,
Received December 21, 1971
Fractionation of nuclear ribonucleic acids (RNA’s) according to molecular size is usually achieved by ultracentrifugation (1,2) or electrophoresis (3,4). These techniques, while yielding a good resolution of polynucleotides with radically different chain lengths, present a number of problems in respect to sample handling, preservation, and recovery, and (excepting very expensive zonal centrifugation equipment) in regard to the practical scale of preparation. The above problems, however, could be partly or fully solved by gel filtration techniques employing highly hydrated large-pore gels, such as the “sphere-condensed” agarose gels (5,6), which have already been applied with some successin fractionation of the nucleic acids (7). We have found that one of the commercially available agarose gels, Sepharose 4B, could effect satisfactory separation of the rapidly sedimenting (3060 S) nuclear ribopolynucleotides from ribosomal RNA’s. MATERIALS
AND
METHODS
Rat liver nuclei were purified by a modified procedure of Chauveau et al. (8) from animals intraperitoneally injected with 6-C14-erotic acid (50 &i/animal), or P3”-orthophosphate (l-2 mCi/animal) , at specified intervals before killing. The purified nuclei, usually from a single liver, were subjected to a limited digestion with chromatographically purified (9) pancreatic DNAse, under conditions described by Penman (10). The DNAse-digested nuclear material was mixed with 2 vol ice-cold 95% (v/v) ethanol, and the precipitated nucleoproteins collected by centrifugation for 10 min at 12,OOOg,,, and 4-6”. Isolation of RNA’s from pelleted nucleoproteins was carried out as described elsewhere (11). The isolated RNA’s were finally dissolved in the filtration solvent (0.05% sodium dodecyl sulfate/O.1 M NaCI/O.Ol M Tris HCl, pH 7.4), and stored frozen until analyzed. The gel filtration 367 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
368
NOVAKOld
AND
PETROVI6
was done on 200 ml beds of Sepharose 4B or 2B (Pharmacia, Uppsala, Sweden), packed in columns of 20-25 mm i.d., with flow rates in the range of 10-30 ml/hr. The recovery of RNA’s from the column fractions was achieved by an ethanol/ether concentration and precipitation. For an approximately threefold concentration from the above solvent, 1.6 vol 95% ethanol and 3.2 vol diethyl ether were added per volume of column effluent. After shaking and phase separation (5-10 min), the lower phase was mixed with 2 vol 95% ethanol, and the precipitation allowed to proceed for 2-3 hr at 0”. Characterization of isolated RNA’s in respect to relative sedimentation rates was performed in linear 1040% (w/v) sucrose gradients, made in the filtration solvent. The gradients were centrifuged for 3 hr at 35,000 rpm in an SW 50L rotor of Spinco model L ultracentrifuge, operated at 20”. The centrifuged gradients were fractionated and analyzed in usual fashion (11). Nonlabeled rat liver ribosomal RNA (0.1 mg) was added to each sample before ultracentrifugation, to serve as an internal sedimentation rate standard. The P32 nucleotide compositions were determined as described (12)) using nonlabeled rat liver ribosomal RNA as a carrier when necessary. Optical density nucleotide compositions were determined by the same method. RESULTS
Figure 1 presents a typical filtration profile of nuclear rapidly labeled RNA’s fractionated on Sepharose 4B. A predominant fraction of the early (20 min) labeling appears in the form of polymers excluded from the gel particles. The sedimentation analysis of this void volume zone material showed a considerable proportion of very rapidly sedimenting polynucleotides, reaching the bottom of sucrose gradients within standard 3 hr sedimentation interval. Treatment with a small amount of pancreatic RNAse quantitatively converts this radioactivity to acid-soluble nucleotides eluting in the region of total column volume (Fig. 1). The UV-absorbing material eluting in the void volume after RNAse treatment represents DNA persisting the DNAse digestion. It is, as seen in Fig. 1, devoid of any newly synthesized deoxypolynucleotides. The filtration profile of the long-term labeled nuclear RNA’s on Sepharose 4B is presented in Fig. 2. RNA’s from the successive filtration zones (starting from the void volume region) were in this case pooled and subjected t.o sucrose gradient centrifugation in the presence of nonlabeled ribosomal RNA as an internal sedimentation rate standard. The void volume region polymers contained more than 60% of
RNA
ISOLATION
10
BY
AGAROSE
20
FRACTION
GEL
FILTRATION
369
30
NUMBER
FIG. 1. Filtration of nuclear rapidly labeled ribonucleic acids on Sepharose 4B. 200 ml column of the gel in SDS/Tris/NaCl buffer; 5 ml fractions. About 7 mg ribosomal RNA was added per 0.3 mg nuclear RNA isolated from liver of animal receiving C”-erotic acid 20 min before hilling: (04) optical density, (@-@) radioactivity in intact sample; (0 - -10) optical density, (0 - - 0) radioactivity in quantitatively identical sample treated with 5 pg pancreatic RNAse before loading to the column (10 min at room temperature).
radioactive ribopolynucleotides sedimenting faster than 29s ribosomal RNA, and little radioactivity sedimenting slower than approximately 20s. The material from further successive zones of the filtration profile consisted of a mixture of the two high molecular weight ribosomal RNA’s, the larger component eluting fast.er than the smaller. This finding was confirmed in several further experiments. As evident from the above data, separation of rapidly sedimenting RNA’s from ribosomal RNA’s could be achieved even in the presence of a large excess of the latter (see especially Fig. 1). In preparative runs, it is convenient to work with up to 100 mg of high molecular weight RNA per liter of gel bed, repeating filtration of the material from the void volume zone in order to remove contaminating ribosomal RNA. This approach was utilized in preparation of rapidly sedimenting RNA for determination of its optical density nucleotide composition (Table 1). As seen in Table 1, the gel-excluded RNA’s contained a high proportion of uridylic and adenylic acids relative to ribosomal RNA recovered from neighboring fractions. The nucleotide compositions of rapidly sedimenting RNA’s isolated by Sepharose 4B gel filtration are reasonably similar to those reported for 305 to 60s nuclear RNA’s isolated by
370
NOVAKOVI6
AND
PETFlOW
60 40 20
12 ,5.0
-IL
3.0
5 E y :: E 8
1.0 8
10
15
20
FRACTION
25
A I
I I
1 1
-=I 0.8
30
NUMBER
FIG. 2. Left: Filtration profile of nuclear uniformly labeled (24 hr with P3Z-orthophosphate) RNA’s on Sepharose 4B. 200 ml column of the gel in SDS/Tris/NaCl buffer; 5 ml fractions. Right: Sedimentation analysis of nuclear RNA’s from zones A, B, and C of agarose filtration run shown in left graph. 0.1 ml aliquots from three successive fractions in indicated areas of Sepharose run were combined and loaded directly on sucrose gradients, which were centrifuged and procesred further as described under “Materials and Methods.” Volume of gradient fractions after dilution for absorbance readings was 0.68 ml.
zonal centrifugation (14,15). However, as seen in Table 1, the optical density nucleotide composition corresponded with the Psa nucleotide composition only for long-term labeled rapidly sedimenting RNA. With Sepharose 2B, separation of the rapidly sedimenting and the ribosomal RNA’s was much less clearcut. On this gel all nuclear ribopolynucleotides had a trailing filtration profile, indicating interaction with the gel matrix. At higher NaCl molarities, the polynucleotides heavier than approximately 205 were selectively retained on Sepharose 2B (13). DISCUSSION
Preparative isolation of nuclear “rapidly sedimenting” or “giant size” RNA’s has been carried out mainly by ultracentrifugation in large horizontal or zonal rotors (14-16). Our results show that these RNA’s could be satisfactorily isolated by Sepharose 4B gel filtration. The method is
RNA
Nucleotide
ISOLATION
Gmpositions
BY
AGAROSE
GEL
TABLE 1 of Nuclear RN.4 Fraction:: Isolated on Sepharose 4B N\lcleotide
8ample
371
FILTRATION
(‘ytidylic acids
Ade Y;ic acids
per cent of LJi,idylic acids
(ha ylic acicis
G + (!, per rent’
40 min labeling, zone “A”’ (P3* composition) 24 hr labeling, zone “A” (Ps2 composition) Zone “A,” optical density composition2 Zone “B,” P32 composition3 Zone “B,” optical densit,y compositiorF 1 Letter designations refer to approximate filtration zones shown in Fig. 2. Thus, zone “A” material consists chiefly of Xl-60s RNA’s, and zone “B” material contains mostl! 29S ribosomal RNA. 2 Data obtained with RNA fractions from a preparative Sepharose 4B run Cth RNA from crude nuclei of five livers. 500 ml column was llsed, and the zone “A” material was concentrated by ethanol/ether precipitation (see “hIaterials and 3Iethods”) and snb.iected to another cycle of Sepharose 4B filtration. Zone “B” material was taken from t.he Erst cycle. 8 24 hr labeling in 1iw with Paz-orthophosphate fabollt 1 mCi/animal\.
very sim;>!e and reLab!e, and does ngt depend on complex or costly equipment. The purity of Sepharose 4B-isolaied rapidly sedimenting RNA’s is better than 60% even at fairly high levels of ribosomal RNA’s, Of cocr_e, tl:c purity could be expecked to vary somewhat, depending on ahsol~~tcq‘antities of RNA loaded on columns, and, to a lower extent, on ratio of ribosom31 and rapidly sedimenting RNA’s in the preparnticn. Rqecl:ed gel filtration could be exI:ected to yield preparations essentially free of figishcd ribosomal RNA, or other monocistronic riboljolynucleotidea. Such preparations could be further fractionated by other methods (l-4,14-16). Overall RNA preservat’ion during isolation was consistently very good in conditions of our experiments. This could be explained by potent inhibition of residual or contaminating nucleodepolymerases by sodium dodecyl sulfate, an anionic detergent capable of strong binding to practically all types of proteins (17,18). Sepharose 4B filtration appears to be useful also for routine characterization of the state of RNA synthesis in eucaryote cells, especially in respect to the relative labeling of nonribosomal and preribosomal versus ribosomal RNA%. Our results for short-term labeled RNA’s (Fig. 1 and Table 1) show that gel-excluded ribopolynucleotides comprise,
372
NOVAKOVIt)
AND
PETROVI6
besides the ribosomal precursor RNA, a high proportion of “giant size” nonribosomal RNA rich in adenylic and uridylic acids. This RNA, known to be present in nuclei from a variety of cell types (1,2,10,1416,19) is difficult to assess by either MAK (methylated albumin-Kieselguhr) chromatography (20) or sedimentation analysis because of its very large molecular size. It appears readily analyzable by means of Sepharose chromatography. SUMMARY
Gel filtration of rat liver nuclear RNA’s on Sepharose 4B permits routine separation of the rapidly sedimenting (30-605) ribopolynucleotides from ribosomal RNA’s. The method could be easily adapted to either analytical characterization or preparative isolation of preribosomal and polycistronic RNA%. ACKNOWLEDGMENTS We acknowledge with thanks the technical assistance of Mrs. Rada Tepavac. This investigation was partly supported by grant 3166/l from Federal Science Council of S.F.R. Yugoslavia to Boris Kidrich Institute. REFERENCES M. MURAMATSU, J. L. HODNETT, W. J. STEELE, AND H. BUSCH, Biochim. Biophys. Acta 123, 116 (1966). 2. J. R. WARNER, R. SOEIRO, H. C. BIRNBOIM, M. GIRARD, AND J. E. DARNELL, J. 1.
Mol.
Biol.
19, 349
(1966).
3. D. H. L. BISHOP, J. R. CLAYBROOK, AND S. SPIEGELMAN, J. Mol. Biol. 26, 373 (1967). 4. C. W. DINCMAN AND A. C. PEACOCK, Biochemistry 7, 659 (1968). 5. S. HJER~~N, B&him. Biophys. Acta 79, 393 (1964). 6. S. BENTSSON AND L. PHILIPSON, Biochim. Biophys. Acta 79, 399 (1964). 7. B. ~BERG AND L. PHILIPSON, Arch. Biochem. Biophys. 119, 504 (1967). 8. J. CHAUVEAU, Y. MOULB, AND CH. ROUILLER, Exp. Cell Res. 11, 317 (1956). 9. J. POLATNICK AND H. L. BACHRACH, Anal. Biochem. 2, 161 (1961). 10. S. PENMAN, J. Mol. Biol. 17, 117 (1966). 11. S. PETROVI~, J. PETROVIE, AND D. KANAZIR, Biochim. Biophys. Acta 119, 213 (1966). 12. S. PETROVI~~ AND B. BRKI~, Biochim. Biophys. Acta 217, 95 (1970). 13. S. PETROVIE, M. NOVAKOVI~, AND J. PETROVI~, Biochim. Biophys. Acta 254, 493 (1971). 14. L. FLOYD, N. OKAMURA, AND H. BUSCH, Biochim. Biophys. Acta 129, 68 (1966). 15. R. SOEIRO, H. C. BIRNBOIM, AND J. E. DARNELL, J. Mol. BioZ. 19, 349 (1966). 16. G. ATTARDI, H. PARNAS, M. HUANG, AND B. ATTARDI, J. Mol. BioZ. 20, 145 (1966). 17. R. PITT-RIVERS AND F. S. A. IMPIOMBATO, Riochem. J. 109, 825 (1968). 18. C. A. NELSON, J. Biol. Chem. 246, 3895 (1971). 19. K. SCHERRER AND L. MARCAUD, Bull. Sot. Chim. BioZ. 47, 1697 (1965). 20. N. YOSHIKAWA-FUKADA, Biochim. Biophys. Actu 123, 91 (1966).
BIOCHEMISTRY
Isolation
of
Nuclear Acids
MILKA Department
367-372 (1972)
49,
Rapidly
by Agarose
B. NOVAKOVIC of Biochemistry,
Sedimenting Gel AND
BOGS Kidlich Beograd, Yugoslavia
Ribonucleic
Filtration S. L. PETROVIC Institute,
P. 0. Box
522,
Received December 21, 1971
Fractionation of nuclear ribonucleic acids (RNA’s) according to molecular size is usually achieved by ultracentrifugation (1,2) or electrophoresis (3,4). These techniques, while yielding a good resolution of polynucleotides with radically different chain lengths, present a number of problems in respect to sample handling, preservation, and recovery, and (excepting very expensive zonal centrifugation equipment) in regard to the practical scale of preparation. The above problems, however, could be partly or fully solved by gel filtration techniques employing highly hydrated large-pore gels, such as the “sphere-condensed” agarose gels (5,6), which have already been applied with some successin fractionation of the nucleic acids (7). We have found that one of the commercially available agarose gels, Sepharose 4B, could effect satisfactory separation of the rapidly sedimenting (3060 S) nuclear ribopolynucleotides from ribosomal RNA’s. MATERIALS
AND
METHODS
Rat liver nuclei were purified by a modified procedure of Chauveau et al. (8) from animals intraperitoneally injected with 6-C14-erotic acid (50 &i/animal), or P3”-orthophosphate (l-2 mCi/animal) , at specified intervals before killing. The purified nuclei, usually from a single liver, were subjected to a limited digestion with chromatographically purified (9) pancreatic DNAse, under conditions described by Penman (10). The DNAse-digested nuclear material was mixed with 2 vol ice-cold 95% (v/v) ethanol, and the precipitated nucleoproteins collected by centrifugation for 10 min at 12,OOOg,,, and 4-6”. Isolation of RNA’s from pelleted nucleoproteins was carried out as described elsewhere (11). The isolated RNA’s were finally dissolved in the filtration solvent (0.05% sodium dodecyl sulfate/O.1 M NaCI/O.Ol M Tris HCl, pH 7.4), and stored frozen until analyzed. The gel filtration 367 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
368
NOVAKOld
AND
PETROVI6
was done on 200 ml beds of Sepharose 4B or 2B (Pharmacia, Uppsala, Sweden), packed in columns of 20-25 mm i.d., with flow rates in the range of 10-30 ml/hr. The recovery of RNA’s from the column fractions was achieved by an ethanol/ether concentration and precipitation. For an approximately threefold concentration from the above solvent, 1.6 vol 95% ethanol and 3.2 vol diethyl ether were added per volume of column effluent. After shaking and phase separation (5-10 min), the lower phase was mixed with 2 vol 95% ethanol, and the precipitation allowed to proceed for 2-3 hr at 0”. Characterization of isolated RNA’s in respect to relative sedimentation rates was performed in linear 1040% (w/v) sucrose gradients, made in the filtration solvent. The gradients were centrifuged for 3 hr at 35,000 rpm in an SW 50L rotor of Spinco model L ultracentrifuge, operated at 20”. The centrifuged gradients were fractionated and analyzed in usual fashion (11). Nonlabeled rat liver ribosomal RNA (0.1 mg) was added to each sample before ultracentrifugation, to serve as an internal sedimentation rate standard. The P32 nucleotide compositions were determined as described (12)) using nonlabeled rat liver ribosomal RNA as a carrier when necessary. Optical density nucleotide compositions were determined by the same method. RESULTS
Figure 1 presents a typical filtration profile of nuclear rapidly labeled RNA’s fractionated on Sepharose 4B. A predominant fraction of the early (20 min) labeling appears in the form of polymers excluded from the gel particles. The sedimentation analysis of this void volume zone material showed a considerable proportion of very rapidly sedimenting polynucleotides, reaching the bottom of sucrose gradients within standard 3 hr sedimentation interval. Treatment with a small amount of pancreatic RNAse quantitatively converts this radioactivity to acid-soluble nucleotides eluting in the region of total column volume (Fig. 1). The UV-absorbing material eluting in the void volume after RNAse treatment represents DNA persisting the DNAse digestion. It is, as seen in Fig. 1, devoid of any newly synthesized deoxypolynucleotides. The filtration profile of the long-term labeled nuclear RNA’s on Sepharose 4B is presented in Fig. 2. RNA’s from the successive filtration zones (starting from the void volume region) were in this case pooled and subjected t.o sucrose gradient centrifugation in the presence of nonlabeled ribosomal RNA as an internal sedimentation rate standard. The void volume region polymers contained more than 60% of
RNA
ISOLATION
10
BY
AGAROSE
20
FRACTION
GEL
FILTRATION
369
30
NUMBER
FIG. 1. Filtration of nuclear rapidly labeled ribonucleic acids on Sepharose 4B. 200 ml column of the gel in SDS/Tris/NaCl buffer; 5 ml fractions. About 7 mg ribosomal RNA was added per 0.3 mg nuclear RNA isolated from liver of animal receiving C”-erotic acid 20 min before hilling: (04) optical density, (@-@) radioactivity in intact sample; (0 - -10) optical density, (0 - - 0) radioactivity in quantitatively identical sample treated with 5 pg pancreatic RNAse before loading to the column (10 min at room temperature).
radioactive ribopolynucleotides sedimenting faster than 29s ribosomal RNA, and little radioactivity sedimenting slower than approximately 20s. The material from further successive zones of the filtration profile consisted of a mixture of the two high molecular weight ribosomal RNA’s, the larger component eluting fast.er than the smaller. This finding was confirmed in several further experiments. As evident from the above data, separation of rapidly sedimenting RNA’s from ribosomal RNA’s could be achieved even in the presence of a large excess of the latter (see especially Fig. 1). In preparative runs, it is convenient to work with up to 100 mg of high molecular weight RNA per liter of gel bed, repeating filtration of the material from the void volume zone in order to remove contaminating ribosomal RNA. This approach was utilized in preparation of rapidly sedimenting RNA for determination of its optical density nucleotide composition (Table 1). As seen in Table 1, the gel-excluded RNA’s contained a high proportion of uridylic and adenylic acids relative to ribosomal RNA recovered from neighboring fractions. The nucleotide compositions of rapidly sedimenting RNA’s isolated by Sepharose 4B gel filtration are reasonably similar to those reported for 305 to 60s nuclear RNA’s isolated by
370
NOVAKOVI6
AND
PETFlOW
60 40 20
12 ,5.0
-IL
3.0
5 E y :: E 8
1.0 8
10
15
20
FRACTION
25
A I
I I
1 1
-=I 0.8
30
NUMBER
FIG. 2. Left: Filtration profile of nuclear uniformly labeled (24 hr with P3Z-orthophosphate) RNA’s on Sepharose 4B. 200 ml column of the gel in SDS/Tris/NaCl buffer; 5 ml fractions. Right: Sedimentation analysis of nuclear RNA’s from zones A, B, and C of agarose filtration run shown in left graph. 0.1 ml aliquots from three successive fractions in indicated areas of Sepharose run were combined and loaded directly on sucrose gradients, which were centrifuged and procesred further as described under “Materials and Methods.” Volume of gradient fractions after dilution for absorbance readings was 0.68 ml.
zonal centrifugation (14,15). However, as seen in Table 1, the optical density nucleotide composition corresponded with the Psa nucleotide composition only for long-term labeled rapidly sedimenting RNA. With Sepharose 2B, separation of the rapidly sedimenting and the ribosomal RNA’s was much less clearcut. On this gel all nuclear ribopolynucleotides had a trailing filtration profile, indicating interaction with the gel matrix. At higher NaCl molarities, the polynucleotides heavier than approximately 205 were selectively retained on Sepharose 2B (13). DISCUSSION
Preparative isolation of nuclear “rapidly sedimenting” or “giant size” RNA’s has been carried out mainly by ultracentrifugation in large horizontal or zonal rotors (14-16). Our results show that these RNA’s could be satisfactorily isolated by Sepharose 4B gel filtration. The method is
RNA
Nucleotide
ISOLATION
Gmpositions
BY
AGAROSE
GEL
TABLE 1 of Nuclear RN.4 Fraction:: Isolated on Sepharose 4B N\lcleotide
8ample
371
FILTRATION
(‘ytidylic acids
Ade Y;ic acids
per cent of LJi,idylic acids
(ha ylic acicis
G + (!, per rent’
40 min labeling, zone “A”’ (P3* composition) 24 hr labeling, zone “A” (Ps2 composition) Zone “A,” optical density composition2 Zone “B,” P32 composition3 Zone “B,” optical densit,y compositiorF 1 Letter designations refer to approximate filtration zones shown in Fig. 2. Thus, zone “A” material consists chiefly of Xl-60s RNA’s, and zone “B” material contains mostl! 29S ribosomal RNA. 2 Data obtained with RNA fractions from a preparative Sepharose 4B run Cth RNA from crude nuclei of five livers. 500 ml column was llsed, and the zone “A” material was concentrated by ethanol/ether precipitation (see “hIaterials and 3Iethods”) and snb.iected to another cycle of Sepharose 4B filtration. Zone “B” material was taken from t.he Erst cycle. 8 24 hr labeling in 1iw with Paz-orthophosphate fabollt 1 mCi/animal\.
very sim;>!e and reLab!e, and does ngt depend on complex or costly equipment. The purity of Sepharose 4B-isolaied rapidly sedimenting RNA’s is better than 60% even at fairly high levels of ribosomal RNA’s, Of cocr_e, tl:c purity could be expecked to vary somewhat, depending on ahsol~~tcq‘antities of RNA loaded on columns, and, to a lower extent, on ratio of ribosom31 and rapidly sedimenting RNA’s in the preparnticn. Rqecl:ed gel filtration could be exI:ected to yield preparations essentially free of figishcd ribosomal RNA, or other monocistronic riboljolynucleotidea. Such preparations could be further fractionated by other methods (l-4,14-16). Overall RNA preservat’ion during isolation was consistently very good in conditions of our experiments. This could be explained by potent inhibition of residual or contaminating nucleodepolymerases by sodium dodecyl sulfate, an anionic detergent capable of strong binding to practically all types of proteins (17,18). Sepharose 4B filtration appears to be useful also for routine characterization of the state of RNA synthesis in eucaryote cells, especially in respect to the relative labeling of nonribosomal and preribosomal versus ribosomal RNA%. Our results for short-term labeled RNA’s (Fig. 1 and Table 1) show that gel-excluded ribopolynucleotides comprise,
372
NOVAKOVIt)
AND
PETROVI6
besides the ribosomal precursor RNA, a high proportion of “giant size” nonribosomal RNA rich in adenylic and uridylic acids. This RNA, known to be present in nuclei from a variety of cell types (1,2,10,1416,19) is difficult to assess by either MAK (methylated albumin-Kieselguhr) chromatography (20) or sedimentation analysis because of its very large molecular size. It appears readily analyzable by means of Sepharose chromatography. SUMMARY
Gel filtration of rat liver nuclear RNA’s on Sepharose 4B permits routine separation of the rapidly sedimenting (30-605) ribopolynucleotides from ribosomal RNA’s. The method could be easily adapted to either analytical characterization or preparative isolation of preribosomal and polycistronic RNA%. ACKNOWLEDGMENTS We acknowledge with thanks the technical assistance of Mrs. Rada Tepavac. This investigation was partly supported by grant 3166/l from Federal Science Council of S.F.R. Yugoslavia to Boris Kidrich Institute. REFERENCES M. MURAMATSU, J. L. HODNETT, W. J. STEELE, AND H. BUSCH, Biochim. Biophys. Acta 123, 116 (1966). 2. J. R. WARNER, R. SOEIRO, H. C. BIRNBOIM, M. GIRARD, AND J. E. DARNELL, J. 1.
Mol.
Biol.
19, 349
(1966).
3. D. H. L. BISHOP, J. R. CLAYBROOK, AND S. SPIEGELMAN, J. Mol. Biol. 26, 373 (1967). 4. C. W. DINCMAN AND A. C. PEACOCK, Biochemistry 7, 659 (1968). 5. S. HJER~~N, B&him. Biophys. Acta 79, 393 (1964). 6. S. BENTSSON AND L. PHILIPSON, Biochim. Biophys. Acta 79, 399 (1964). 7. B. ~BERG AND L. PHILIPSON, Arch. Biochem. Biophys. 119, 504 (1967). 8. J. CHAUVEAU, Y. MOULB, AND CH. ROUILLER, Exp. Cell Res. 11, 317 (1956). 9. J. POLATNICK AND H. L. BACHRACH, Anal. Biochem. 2, 161 (1961). 10. S. PENMAN, J. Mol. Biol. 17, 117 (1966). 11. S. PETROVI~, J. PETROVIE, AND D. KANAZIR, Biochim. Biophys. Acta 119, 213 (1966). 12. S. PETROVI~~ AND B. BRKI~, Biochim. Biophys. Acta 217, 95 (1970). 13. S. PETROVIE, M. NOVAKOVI~, AND J. PETROVI~, Biochim. Biophys. Acta 254, 493 (1971). 14. L. FLOYD, N. OKAMURA, AND H. BUSCH, Biochim. Biophys. Acta 129, 68 (1966). 15. R. SOEIRO, H. C. BIRNBOIM, AND J. E. DARNELL, J. Mol. BioZ. 19, 349 (1966). 16. G. ATTARDI, H. PARNAS, M. HUANG, AND B. ATTARDI, J. Mol. BioZ. 20, 145 (1966). 17. R. PITT-RIVERS AND F. S. A. IMPIOMBATO, Riochem. J. 109, 825 (1968). 18. C. A. NELSON, J. Biol. Chem. 246, 3895 (1971). 19. K. SCHERRER AND L. MARCAUD, Bull. Sot. Chim. BioZ. 47, 1697 (1965). 20. N. YOSHIKAWA-FUKADA, Biochim. Biophys. Actu 123, 91 (1966).