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Separation of ribosomal RNAs on agarose gels
BIOCHIMICAET BIOPHYSICAACTA
493
BBA Report BBA 91335 S e p a r a t i o n o f r i b o s o m a l R N A s on agarose gels
SVETOZAR PETROVIC, MILKA NOVAKOVICand JELENA PETROV1C Department o f Biochemistry, Boris Kidrich Institute, P.O. Box 522, Beograd (Yugoslavia)
(Received October 22nd, 1971)
SUMMARY
28-S ribosomal RNA of rat liver is selectively retained in highly hydrated 'sphere-condensed' agarose gels equilibrated at 21-25 ° with a sodium dodecyl sulfate-Tris-EDTA buffer containing 0.5 M NaC1. The adsorbed or gelated polynucleotide could be recovered by elution with 0.1 M NaC1 in the same buffer, or by raising the temperature to 35 °. The density gradient profiles and nucleotide compositions indicate that the separation under the described conditions is close to being quantitiative.
Agarose gels of highly open particle structure 1, 2 could effect a molecular sieving separation of polynucleotides radically differing in molecular weight 3- s. With polynucleotides of less different molecular weights, such as the two high molecular weight rRNAs, interactions of the polymers with each other and with the gel matrix s interfere with the separation. Attempting to improve the separation by increasing the ionic strength of the filtration buffer, we have observed that the larger component of liver rRNA tends to be selectively retained on agarose at higher ionic strength. At an NaC1 molarity of approximately 0.5, it was possible to achieve a clear-cut separation of 18-S and 4-6-S polymers from 28-S rRNA, which undergoes a reversible adsorption, possibly associated with thermal gelation. RNAs were isolated 6 from liver microsomes of the Belgrade variant of Wistar rats. The solution of purified 7 ribopolynucleotides in 0.5 M NaC1-0.1% sodium dodecyl sulfate-0.0025 M EDTA (sodium salt), pH 7.5-0.02 M Tris-HC1, pH 7.5, was kept frozen until used. Sepharose 2B or 4B (Pharmacia, Uppsala) was packed in jacketed glass columns; 50-200-ml beds were used in the range of 2.5-10 mg RNA. The gels were carefully equilibrated with the above solvent before packing. All column operations were carried out at temperatures above 21 °, because of the tendency of the detergent to salt out at the relatively high salt concentration employed. Also, some adsorption of the smaller rRNA was apparent at 20-21 °, especially with Sepharose 4B. Concentration of RNA in Biochim. Biophy~ Acta, 254 (1971)493-495
494
BBA REPORT
the range of 1-5 mg/ml did not influence the separation. Elution was carried out at 23-25 °, with a flow rate of 5-10 ml/h per 100 ml gel. Higher flow rates or temperatures resulted in elution of increasing amounts of 28-S RNA together with 18-S material. After elution of 18-S and 4-6-S RNAs, 28-S RNA was recovered by changing the molarity of NaCI in sodium dodecyl sulfate-Tris-EDTA buffer to 0.1 M. Slightly more than one column volume of this buffer was needed for a complete elution of the larger rRNA (Fig. 1). The nucleic acids were recovered from the eluates by an ethanol-ether concentration and precipitation 5 , and characterized by density gradient centrifugation (see the legend to Fig. 1).
60I ~0
40 20
lo
<: 1.0
' lo 20 FRACTION NUMBER 25
COLUMN VOLUMES
Fig. 1. (Left) Fractionation of 6.3 mg of rat liver rRNA on a 200-ml bed of Sepharose 2B. The elution was with 0.5 M NaCI in sodium dodecyl sulfate~Tris-EDTA buffer (see the text) and then with 0.1 M NaCI in the same buffer. (Right) Density gradient characterization of ether-ethanol precipitated RNAs from the Sepharose 2B chromatography run described on the left graph. The materials recovered from Peaks A and B were dissolved in equal volumes of 0.1 M NaCI in sodium dodecyl sulfate-Tds-EDTA buffer and equal aliquots centrifuged for 3 h at 35 000 rev./min in linear 10-40% sucrose gradients made in the same buffer. The centrifugation was done at 20° . Direction of sedimentation from right to left.
TABLE I NUCLEOTIDE COMPOSITIONS OF rRNAs ISOLATED BY SEPHAROSE 2B GEL CHROMATOGRAPHY
G+C (mole %)
Moles/ l O0 moles o f nucleotide
18-S RNA* 28-S RNA
Cytidylic acid
Adenylic acid
Uridylic acid
Guanylic acid
25.7 32.4
22.7 16.6
19.8 15.7
31.8 35.3
*This material was freed from 4-6-S RNA by gel filtration on Sepharose 4B s
Biochira. Biophy~ Acta, 254 (1971) 493-495
57.5 67.7
BBA REPORT
495
Fig. 1 presents the results of the standard separation procedure described above. As seen from the sucrose gradient profiles of separated RNAs (right graph), the larger rRNA did not contain detectable amounts of 18-S RNA, and vice versa. The analysis of the nucleotide compositions (Table I) revealed significant differences between the two rRNAs, especially in overall G+C content. In this respect, our analysis gave results almost indistinguishable from those of Hirsch 9 . Experiments at elevated temperatures revealed that the extent of adsorption of the larger rRNA progressively diminishes as the temperature rises. At 35 °, essentially all rRNA elutes within a single bed volume with 0.5 M NaCI in sodium dodecyl sulfate-TrisEDTA buffer. This observation seems to support the idea that the established separation results, at least in part, from preferential thermal gelation of the larger rRNA under conditions of high ionic strength. Examination of Sephadex gel filtration media as possible separation vehicles for rRNAs gave negative results. Both rRNAs were completely excluded from Sephadex G-100 and G-200 (Pharmacia, Uppsala), regardless of the molarity of NaC1 in sodium dodecyl sulfate-Tris-EDTA buffer. Thus, the nature of the gel matrix, and hence some chromatographic affinity, could be important for separation. However, it is also possible that penetration into gel particles is necessary for either gelation or chromatographic adsorption of the larger amounts of ribopolynucleotides. The elucidation of the precise mechanisms underlying the observed separation obviously requires further study. However, it is clear that the described chromatographic procedure should be applicable to essentially all mammalian rRNAs, as well as to rRNAs of other vertebrates and eucaryotes. REFERENCES 1 2 3 4 5 6 7 8 9
S. Hjert6n, Biochim. Biophys. Acta, 79 (1964) 393. S..Bengtsson and L. Philipson, Biochim. Biophys. Acta, 79 (1964) 399. B. Oberg and L. Philipson, Arch. Biochem. Biophys., 119 (1967) 504. R. Falcoff and E. Falcoff, Biochim. Biophys, Acta, 199 (1970) 147. M. Novakovid, S. Petrovid and J. Petrovid,Anal. Biochem., submitted for publication. S. Petrovid, J. Petrovid and D. Kanazir, Biochim. Biophys. Acta, 119 (1966) 213. J. Petrovid and S. Petrovid,AnaL Biochern., 15 (1966) 187. S. Petrovid and V. Jankovid, Bull. B. Kidrich Inst. Nucl. ScL Beograd, 13 (1962) 47. C.A. Hirsch, Biochirn. Biophys. Acta, 123 (1966) 246.
Biochim. Biophys. Acta, 254 (1971) 4 9 3 - 4 9 5
493
BBA Report BBA 91335 S e p a r a t i o n o f r i b o s o m a l R N A s on agarose gels
SVETOZAR PETROVIC, MILKA NOVAKOVICand JELENA PETROV1C Department o f Biochemistry, Boris Kidrich Institute, P.O. Box 522, Beograd (Yugoslavia)
(Received October 22nd, 1971)
SUMMARY
28-S ribosomal RNA of rat liver is selectively retained in highly hydrated 'sphere-condensed' agarose gels equilibrated at 21-25 ° with a sodium dodecyl sulfate-Tris-EDTA buffer containing 0.5 M NaC1. The adsorbed or gelated polynucleotide could be recovered by elution with 0.1 M NaC1 in the same buffer, or by raising the temperature to 35 °. The density gradient profiles and nucleotide compositions indicate that the separation under the described conditions is close to being quantitiative.
Agarose gels of highly open particle structure 1, 2 could effect a molecular sieving separation of polynucleotides radically differing in molecular weight 3- s. With polynucleotides of less different molecular weights, such as the two high molecular weight rRNAs, interactions of the polymers with each other and with the gel matrix s interfere with the separation. Attempting to improve the separation by increasing the ionic strength of the filtration buffer, we have observed that the larger component of liver rRNA tends to be selectively retained on agarose at higher ionic strength. At an NaC1 molarity of approximately 0.5, it was possible to achieve a clear-cut separation of 18-S and 4-6-S polymers from 28-S rRNA, which undergoes a reversible adsorption, possibly associated with thermal gelation. RNAs were isolated 6 from liver microsomes of the Belgrade variant of Wistar rats. The solution of purified 7 ribopolynucleotides in 0.5 M NaC1-0.1% sodium dodecyl sulfate-0.0025 M EDTA (sodium salt), pH 7.5-0.02 M Tris-HC1, pH 7.5, was kept frozen until used. Sepharose 2B or 4B (Pharmacia, Uppsala) was packed in jacketed glass columns; 50-200-ml beds were used in the range of 2.5-10 mg RNA. The gels were carefully equilibrated with the above solvent before packing. All column operations were carried out at temperatures above 21 °, because of the tendency of the detergent to salt out at the relatively high salt concentration employed. Also, some adsorption of the smaller rRNA was apparent at 20-21 °, especially with Sepharose 4B. Concentration of RNA in Biochim. Biophy~ Acta, 254 (1971)493-495
494
BBA REPORT
the range of 1-5 mg/ml did not influence the separation. Elution was carried out at 23-25 °, with a flow rate of 5-10 ml/h per 100 ml gel. Higher flow rates or temperatures resulted in elution of increasing amounts of 28-S RNA together with 18-S material. After elution of 18-S and 4-6-S RNAs, 28-S RNA was recovered by changing the molarity of NaCI in sodium dodecyl sulfate-Tris-EDTA buffer to 0.1 M. Slightly more than one column volume of this buffer was needed for a complete elution of the larger rRNA (Fig. 1). The nucleic acids were recovered from the eluates by an ethanol-ether concentration and precipitation 5 , and characterized by density gradient centrifugation (see the legend to Fig. 1).
60I ~0
40 20
lo
<: 1.0
' lo 20 FRACTION NUMBER 25
COLUMN VOLUMES
Fig. 1. (Left) Fractionation of 6.3 mg of rat liver rRNA on a 200-ml bed of Sepharose 2B. The elution was with 0.5 M NaCI in sodium dodecyl sulfate~Tris-EDTA buffer (see the text) and then with 0.1 M NaCI in the same buffer. (Right) Density gradient characterization of ether-ethanol precipitated RNAs from the Sepharose 2B chromatography run described on the left graph. The materials recovered from Peaks A and B were dissolved in equal volumes of 0.1 M NaCI in sodium dodecyl sulfate-Tds-EDTA buffer and equal aliquots centrifuged for 3 h at 35 000 rev./min in linear 10-40% sucrose gradients made in the same buffer. The centrifugation was done at 20° . Direction of sedimentation from right to left.
TABLE I NUCLEOTIDE COMPOSITIONS OF rRNAs ISOLATED BY SEPHAROSE 2B GEL CHROMATOGRAPHY
G+C (mole %)
Moles/ l O0 moles o f nucleotide
18-S RNA* 28-S RNA
Cytidylic acid
Adenylic acid
Uridylic acid
Guanylic acid
25.7 32.4
22.7 16.6
19.8 15.7
31.8 35.3
*This material was freed from 4-6-S RNA by gel filtration on Sepharose 4B s
Biochira. Biophy~ Acta, 254 (1971) 493-495
57.5 67.7
BBA REPORT
495
Fig. 1 presents the results of the standard separation procedure described above. As seen from the sucrose gradient profiles of separated RNAs (right graph), the larger rRNA did not contain detectable amounts of 18-S RNA, and vice versa. The analysis of the nucleotide compositions (Table I) revealed significant differences between the two rRNAs, especially in overall G+C content. In this respect, our analysis gave results almost indistinguishable from those of Hirsch 9 . Experiments at elevated temperatures revealed that the extent of adsorption of the larger rRNA progressively diminishes as the temperature rises. At 35 °, essentially all rRNA elutes within a single bed volume with 0.5 M NaCI in sodium dodecyl sulfate-TrisEDTA buffer. This observation seems to support the idea that the established separation results, at least in part, from preferential thermal gelation of the larger rRNA under conditions of high ionic strength. Examination of Sephadex gel filtration media as possible separation vehicles for rRNAs gave negative results. Both rRNAs were completely excluded from Sephadex G-100 and G-200 (Pharmacia, Uppsala), regardless of the molarity of NaC1 in sodium dodecyl sulfate-Tris-EDTA buffer. Thus, the nature of the gel matrix, and hence some chromatographic affinity, could be important for separation. However, it is also possible that penetration into gel particles is necessary for either gelation or chromatographic adsorption of the larger amounts of ribopolynucleotides. The elucidation of the precise mechanisms underlying the observed separation obviously requires further study. However, it is clear that the described chromatographic procedure should be applicable to essentially all mammalian rRNAs, as well as to rRNAs of other vertebrates and eucaryotes. REFERENCES 1 2 3 4 5 6 7 8 9
S. Hjert6n, Biochim. Biophys. Acta, 79 (1964) 393. S..Bengtsson and L. Philipson, Biochim. Biophys. Acta, 79 (1964) 399. B. Oberg and L. Philipson, Arch. Biochem. Biophys., 119 (1967) 504. R. Falcoff and E. Falcoff, Biochim. Biophys, Acta, 199 (1970) 147. M. Novakovid, S. Petrovid and J. Petrovid,Anal. Biochem., submitted for publication. S. Petrovid, J. Petrovid and D. Kanazir, Biochim. Biophys. Acta, 119 (1966) 213. J. Petrovid and S. Petrovid,AnaL Biochern., 15 (1966) 187. S. Petrovid and V. Jankovid, Bull. B. Kidrich Inst. Nucl. ScL Beograd, 13 (1962) 47. C.A. Hirsch, Biochirn. Biophys. Acta, 123 (1966) 246.
Biochim. Biophys. Acta, 254 (1971) 4 9 3 - 4 9 5