- Email: [email protected]
[79] Preparation of highly purified sRNA from yeast
[78]
AMI~'OACYL SOLUBLE RNA ON SEPHADEX
601
line). The fractions containing purified 14C-leucyl sRNA are pooled, 2 volumes of cold (4 ° ) ethanol are added, and the precipitate is collected by centrifugation at 2000 g for 10 minutes. The precipitated purified 14C-leucyl-sRNA is dissolved in 5-10 ml of water and then dialyzed against two changes (1 liter each, 2 hours) of water; the material is conveniently stored frozen in 1-ml aliquots at --14 °. The specific radioactivity of this product was 136,000 cpm per milligram of RNA. Characterization of R N A Fractions EIuted. Purified ~4C-leucyl-sRNA, obtained as shown in Fig. 1 (fractions 27-35), was analyzed by rechromatography on Sephadex G-200 (Fig. 2) and by sucrose gradient centrifugation (Fig. 3). Both procedures indicate that the preparation is free of high molecular weight RNA. / I I' I I I I 3'0 ~ ~ l j
~
I
2.0 1.0 a~
"~
I
O0
I0
20
0
I
I0
•~BOTTOM
I
i
i
20 TOP ~,
FRACTION NUMBER
Fro. 4. Sucrose gradient analysis of (A) high-molecular weight RNA obtained from the "pH 5 enzymes" fraction by gel filtration, as shown in Fig. 1 (fractions 12-19) and (B) rat liver ribosomal RNA. Approximately 1 mg of (A) and 1.9 mg of (B) in 1 ml of 1% potassium acetate, pH 4.6, were centrifuged on sucrose gradients as described in Fig. 3. Sucrose gradient centrifugation of the high molecular weight RNA, obtained as shown in Fig. 1 (fractions 12-19), reveals two components {Fig. 4A) exhibiting sedimentation coefficients similar to those of ribosomal RNA shown here for comparison (Fig. 4B).
[79] P r e p a r a t i o n of H i g h l y P u r i f i e d s R N A f r o m Y e a s t
By TOMAS LINDAHL and JACQUES R. FRESCO A method for the purification of large quantities of unfractionated sRNA from yeast is described. The product is essentially free from contaminants and is very stable.
602
ISOLATION AND FRACTIONATION* OF NUCLEIC ACIDS
[79]
The starting material is sRNA prepared according to Holley, 1 or commercially available sRNA of good quality, sRNA prepared from cells frozen and thawed before the initial phenol extraction often contains large amounts of contaminating substances difficult to remove and should be avoided. Preparation Principle. After an initial ethanol precipitation of the crude sRNA, the preparation is extracted with phenol to remove traces of nuclease activity and denatured proteins. Ammonium sulfate is added to precipitate high molecular weight contaminants, leaving sRNA in solution; the sRNA is recovered by precipitation with a higher concentration of ammonium sulfate. Dialysis against EDTA removes polyvalent cations, and prolonged dialysis against sodium chloride and water gives a preparation free from contaminating small molecules. The material is finally freezedried and stored as a solid. Smith et al. 2 reported that an RNA fraction incapable of accepting amino acids is precipitated at low ammonium sulfate concentrations. Accordingly, a purification step of this type has been used to remove high molecular weight RNA as well as other macromolecular contaminants seen as fast-moving components in sedimentation velocity experiments in the analytical ultracentrifuge. A small proportion of the sRNA coprecipitates with the high molecular weight material, but without significant fractionation of the different amino acid specific sRNA's (Fig. 1). All other purification steps give essentially quantitative recovery of sRNA. A small amount of larger nucleic acid components sometimes remains in the sRNA, depending on the effectiveness of the ammonium sulfate fractionation step. If complete absence of such material is necessary, it can be removed more reproducibly by substituting a gel filtration step for the ammonium sulfate fractionation. Whereas the ammonium sulfate step is easily applicable to preparations on a very large scale, it is inconvenient to similarly scale-up the gel filtration step. Reagents
Tris-hydroehloride, 1 M, pH 7.5 NaC1, 1 M Ethanol, 95% EDTA, sodium salt, 0.1 M, pH 7.0 1R. W. Holley, Biochem. Biophys. Res. Commun. 10, 186 (1963). 2K. C. Smith, E. Cordes, and R. S. Schweet, Biochim. Biophys. Acta 33, 286 (1959).
[79]
SOLUBLE RNA
YEAST
603
SALTING OUT OF Y E A S T s-RNA F R O M W A T E R WITH AMMONIUM SULFATE. RNA-CONCENTRATION 5mg/ml --I
I00
I
I
I
I
90 ~
~-
I v I I . . . . . . . . . . ~..__~
I00
r"
o~ o ~
90
:
o -,..x° 7o
~.: ......
,/ . . ~ < ; " i: .......... 4.."~!..-,!.-A
80 __
p
oPER.AE \
sc
oo X / , %::/
E 5C O
' ~// /o ../~
t",J 4C
PRECIPITATE
W':/
~:.:,.--o
L:J: /
7o o
z
o,'.,'
6o x
z£" .i
/ ¢J
80:1. E
• VALtNE . . . . . . . . "TYROSINE. . . . . • BERINE ..........
o ALANiNE ........
50 ~> - -
40 (~
9t
20
20 % .
,o
• ,.,"
%
o
oZ.',.~ ..... 40
45
50
55
60
65
,o 70
75
80.
85
90
% AMMONIUM SULFATE SATURATION
FIG. 1. Salting out of sRNA with ammonium sulfate. Different amounts of ammonium sulfate were added to aliquots of an sRNA solution. After 20 hours at 4 °, the mixtures were centrifuged in the cold, and A ~ of the supernatants was measured. The precipitates were dissolved in buffer solution, and A~0 and amino acid acceptor activity were determined. Solid lines refer to A~0, dotted and dashed lines to total activity (A~o × amino acid acceptor activity, where acceptor activity of unfractionated sRNA is taken as 1.0). The precipitate formed at 45% ammonium sulfate saturation was recovered, dialyzed against distilled H:O, and subjected to a second complete salting-out experiment. The small but significant amount of valyl-acceptor RNA in this material, which is normally discarded, precipitated mainly between 55% and 70% ammonium sulfate saturation, as does the bulk of valyl-sRNA. This result indicates that nonspecific coprecipitation of sRNA, rather than a selective removal of certain sl%NA molecules, occurs at 45% ammonium sulfate saturation.
Phenol, freshly redistilled, water-saturated (at room temperature) Diethyl ether, A.R., (peroxide free), water-saturated Ammonium sulfate, A.R., pH of a 5% solution----5.2- 5.4 Dialysis tubing, Visking size 20 D.C. The tubing is immersed for 24 hours in 0.01 M Na~-EDTA, pH 7.0, and then boiled for 15 minutes in each of three changes of distilled water. Sephadex G-100 (Pharmacia Fine Chemicals, Inc.). The gel is added slowly under vigorous stirring to a large volume of 0.05 M potassium phosphate, pH 6.8, and boiled for 3 hours. After 10 minutes the gel has settled and the supernatant is decanted; hot distilled water (60-80 °) is added, and the gel is resuspended. The gel is washed with hot water four more times, then at room temperature several times with 1 M NaC1 + 0.01 M Tris-HC1 +
604
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[79]
0.001 M EDTA, pH ~--7.5. Immediately before packing, the gel suspension is transferred to a large suction flask and briefly deaerated under vacuum. Procedure. Five grams of crude sRNA is dissolved in 1000 ml of 0.2 M NaC1 ~ 0.01 M Tris pH ~ 7.5; 1500 ml of cold ethanol is slowly added to the solution with stirring, and the preparation is left overnight at 4 °. The supernatant is decanted and discarded; the precipitate is recovered by low speed centrifugation and washed once with 8 0 ~ ethanol, once with 95% ethanol, and dried in v a c u o over P205 at room temperature. The dry residue is dissolved in 200 ml of 0.001 M EDTA, pH ~ 7.0 and shaken vigorously with an equal volume of phenol for 10 minutes at room temperature. The emulsion is chilled and the phases are separated by low speed centrifugation in the cold. The top (aqueous) layer is removed by aspiration and saved. The remaining material is briefly extracted with 50 ml 0.001 M EDTA, and after low-speed centrifugation the small clear top layer is recovered by aspiration, taking care to avoid contamination with interphase material. The combined aqueous layers are briefly extracted four times with ether; residual ether in the aqueous phase is removed by bubbling filtered nitrogen through the solution. Solid ammonium sulfate is slowly added with stirring at room temperature in the proportion of 250 g to 1000 ml RNA solution (43~ saturation at 4°). The solution is then left at 4 ° for 20 hours, during which time a precipitate slowly develops. The precipitate is discarded after low speed centrifugation in the cold. More ammonium sulfate, 360 g to 1000 ml RNA solution (final concentration, 95% saturation at 4°), is added to the supernatant, and after 16-20 hours at 4 ° the precipitate is recovered by low speed centrifugation and suspended in 20-30 ml distilled H20. The sRNA is dialyzed, first against two changes of 50-100 volumes of 0.2 M NaC1 ~ 0.05 M EDTA, pH ~--7.0 at 4 ° over 24 hours, then against 3 changes of 1 M NaC1 for 48 hours, and finally against 5 changes of distilled H20 over 30 hours. Efficient dialysis is achieved by agitating the dialyzate. Extensive stretching of the dialysis sac can occur during the dialysis against H20, resulting in slight permeability of the membrane to sRNA2 The losses resulting thereby may be avoided by replacing the sac once or twice during this step. sRNA is recovered from the clear solution by freeze-drying. The white, fluffy solid is rapidly transferred to glass vials with air-tight 3j. Goldstein a n d L. C. Craig, J. A m . Chem. Sac. 82, 1833 (1960).
[79]
YEAST SOLUBLE RNA
605
screw caps and stored at room temperature or 4 °. The yield is 2.5-4.0 g, depending on the purity of the starting material. Alter~ative Procedure. After the initial ethanol precipitation and phenol treatment, the dry residue is dissolved in 80 ml of 1 M NaC1 Jr 0.01 M Tris-HC1-}- 0.001 M EDTA, pH ~7.5, heated at 40 ° for 30 minutes, and cooled to room temperature. The heating step releases some sRNA trapped as aggregates or in inactive conformations. If the preparation contains high-molecular weight RNA, a small precipitate can develop which should be removed by low-speed centrifugation. The sRNA solution is then applied to a column of Sephadex G-100 (5)< 160 cm), equilibrated with the same solvent at room temperature. The eluant is
400--
TyrAIo
300200 --
~ii
A258
S
100- Kd=O~/ 0 lOOO 1300I 700
Phe/
/
1 ~' 1600
1900
Kd=l$ T 22oo
EFFLUENT (ml)
Fro. 2. Sephadex G-100 gel filtration of crude.yeast sRNA. The fractions within the main peak were assayed for alanine-, phenylalanine-, serine-, tyrosine-, and valine-acceptor activities; the peak fraction for each activity is indicated. K~---distribution coefficient. passed through the column under a hydrostatic pressure of 50 cm. The effluent is collected in 15-20 ml fractions, and the ultraviolet absorption of each fraction is measured. A typical elution profile is shown in Fig. 2. The material appearing before the main peak consists of aggregated sRNA as well as ribosomal RNA (degraded high-molecular weight rRNA and 5 S rRNA). Some fractionation of the different amino acid-specific sRNA molecules occurs within the main peak; thus, the phenylalanineand serine-accepting molecules appear on the ascending slope. It is therefore important to recover the entire main peak if fractionation of the sRNA is to be avoided. The fractions within the peak are pooled, and the sRNA is precipitated and washed with ethanol as earlier. The precipitate is suspended in 20-30 ml distilled H20, and dialyzed and freeze-dried as above.
606
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[79]
Properties 4 The amino acid acceptor activity per milligram of the purified s R N A is 1.2-]..8 times t h a t of the starting material. The preparation has a phosphorus content of 9.0%. After exhaustive hydrolysis by pancreatic RNase, the only nucleosides observed (thin-layer chromatography) are cytidine and adenosine. The content of protein (biuret) is < 0 . 5 % , D N A < 0.1%, and starch (as iodine-reactive material) < 0 . 1 % . The product is free from nuclease activity and can be kept in solution at 20 ° for 48 hours without decrease in amino acid acceptor activity or molecular PROPERTIES OF YEAST s R N A a,b
Data obtained in
Parameter 22° AX% e(P) M~ (osmotic pressure) M~ (sedimentation equilibrium) M~ (sedimentation equilibrium) Partial specific volume (after dialysis against excess solvent) S~0.~
0.2 M NaC1 -{- 0.01 M phosphate (Na+) -{0.0005 M EDTA, pH 6.85
0.15 M KC1% 0.01 M cacodylate (K+) + 0.005 M MgC12 -t0.0005 M EDTA, pH 7.0
215 7400 26,500 _ 300 26,300 _+ 300
209 7200 ---
27,400 ± 600
--
0.531 ± 0.002 ml/g 4 . 0 0 ± 0 . 0 5 S ( a t 2 0 °)
0.505 ± 0.002 ml/g 4 . 1 0 ± 0 . 0 5 S ( a t 3 0 °)
a Values for sRNA largely devoid of terminal adenosine residue. b Ultraviolet optics have been used in experiments with the analytical ultracentrifuge. weight (if plastic gloves and carefully cleaned glassware have been used in the preparative work in order to minimize contamination with "finger nuclease"5). The w a t e r content of the freeze-dried material is 6-10%. After storage at room t e m p e r a t u r e for 6 months, no change was observed in biological activity or physical chemical solution properties. D r y i n g over P20s at room t e m p e r a t u r e reduces the w a t e r content to ~ 2 % without 4T. Lindahl, D. D. Henley, and J. R. Fresco, J. A m . Chem. Soc. 87, 4961 (1965); D. D. Henley, T. Lindahl, and J. R. Fresco, Proc. Natl. Acad. Sci. U.S. 55, 191 (lo66). ~R. W. Holley, J. Apgar, and S. H. Merrill, J. Biol. Chem. 236, PC42 (1961).
[80]
MAMMALIAN R~A
607
loss of biological activity. Solid RNA is very hygroscopic and should be kept protected from air. Some physical properties of the purified material are given in the table. Acknowledgment This work was supported by grant~ from the National Science Foundation (GB-492), the National Institutes of Health (GM-07654) and the American Heart Association.
[80] Preparation
of R N A
from Mammalian
Ribosomes
By Klvm MOLDAVE
Rat liver ribosomes, purified by several washes in solutions containing high concentrations of magnesium ions as described in this volume [56a], are extracted with a mixture of sodium dodecyl sulfate and watersaturated phenol. The RNA is recovered from the aqueous phase by precipitation with ethanol, and then the high molecular weight ribosomal RNA is resolved from low molecular weight sRNA by molecular sieve chromatography. 1 Reagents
2-Amino-2-hydroxymethylpropane-l,3-diol (Tris)-HC1 buffer, 0.01 M, pH 7.4 Sodium dodecyl sulfate, 3%, in 0.01 M Tris buffer, pH 7.4 Freshly prepared water-saturated phenol (at 4 ° ) Potassium acetate, 20%, adjusted to pH 5.0 with acetic acid Potassium acetate, 20%, pH 5.0 Extraction o] Nucleic Acids. Approximately 50 mg of purified ribosomes ~ are resuspended by gentle homogenization in 20 ml of 0.01 M Tris-HC1 buffer, pH 7.4. An equal volume of 3% sodium dodecyl sulfate is added, and the suspension is stirred with a magnetic mixer for 10 minutes at room temperature. Forty milliliters of water-saturated phenol is then added and the mixture is stirred vigorously for 1 hour at 4 °. All subsequent steps are carried out at 4 °. The resulting emulsion is centri-
1R. P. Sutter and K. Moldave, J. Biol. Chem. 241, 1698 (1966). 'E. Gasior and K. Moldave, J. Biol. Chem. 240, 3346 (1965).
AMI~'OACYL SOLUBLE RNA ON SEPHADEX
601
line). The fractions containing purified 14C-leucyl sRNA are pooled, 2 volumes of cold (4 ° ) ethanol are added, and the precipitate is collected by centrifugation at 2000 g for 10 minutes. The precipitated purified 14C-leucyl-sRNA is dissolved in 5-10 ml of water and then dialyzed against two changes (1 liter each, 2 hours) of water; the material is conveniently stored frozen in 1-ml aliquots at --14 °. The specific radioactivity of this product was 136,000 cpm per milligram of RNA. Characterization of R N A Fractions EIuted. Purified ~4C-leucyl-sRNA, obtained as shown in Fig. 1 (fractions 27-35), was analyzed by rechromatography on Sephadex G-200 (Fig. 2) and by sucrose gradient centrifugation (Fig. 3). Both procedures indicate that the preparation is free of high molecular weight RNA. / I I' I I I I 3'0 ~ ~ l j
~
I
2.0 1.0 a~
"~
I
O0
I0
20
0
I
I0
•~BOTTOM
I
i
i
20 TOP ~,
FRACTION NUMBER
Fro. 4. Sucrose gradient analysis of (A) high-molecular weight RNA obtained from the "pH 5 enzymes" fraction by gel filtration, as shown in Fig. 1 (fractions 12-19) and (B) rat liver ribosomal RNA. Approximately 1 mg of (A) and 1.9 mg of (B) in 1 ml of 1% potassium acetate, pH 4.6, were centrifuged on sucrose gradients as described in Fig. 3. Sucrose gradient centrifugation of the high molecular weight RNA, obtained as shown in Fig. 1 (fractions 12-19), reveals two components {Fig. 4A) exhibiting sedimentation coefficients similar to those of ribosomal RNA shown here for comparison (Fig. 4B).
[79] P r e p a r a t i o n of H i g h l y P u r i f i e d s R N A f r o m Y e a s t
By TOMAS LINDAHL and JACQUES R. FRESCO A method for the purification of large quantities of unfractionated sRNA from yeast is described. The product is essentially free from contaminants and is very stable.
602
ISOLATION AND FRACTIONATION* OF NUCLEIC ACIDS
[79]
The starting material is sRNA prepared according to Holley, 1 or commercially available sRNA of good quality, sRNA prepared from cells frozen and thawed before the initial phenol extraction often contains large amounts of contaminating substances difficult to remove and should be avoided. Preparation Principle. After an initial ethanol precipitation of the crude sRNA, the preparation is extracted with phenol to remove traces of nuclease activity and denatured proteins. Ammonium sulfate is added to precipitate high molecular weight contaminants, leaving sRNA in solution; the sRNA is recovered by precipitation with a higher concentration of ammonium sulfate. Dialysis against EDTA removes polyvalent cations, and prolonged dialysis against sodium chloride and water gives a preparation free from contaminating small molecules. The material is finally freezedried and stored as a solid. Smith et al. 2 reported that an RNA fraction incapable of accepting amino acids is precipitated at low ammonium sulfate concentrations. Accordingly, a purification step of this type has been used to remove high molecular weight RNA as well as other macromolecular contaminants seen as fast-moving components in sedimentation velocity experiments in the analytical ultracentrifuge. A small proportion of the sRNA coprecipitates with the high molecular weight material, but without significant fractionation of the different amino acid specific sRNA's (Fig. 1). All other purification steps give essentially quantitative recovery of sRNA. A small amount of larger nucleic acid components sometimes remains in the sRNA, depending on the effectiveness of the ammonium sulfate fractionation step. If complete absence of such material is necessary, it can be removed more reproducibly by substituting a gel filtration step for the ammonium sulfate fractionation. Whereas the ammonium sulfate step is easily applicable to preparations on a very large scale, it is inconvenient to similarly scale-up the gel filtration step. Reagents
Tris-hydroehloride, 1 M, pH 7.5 NaC1, 1 M Ethanol, 95% EDTA, sodium salt, 0.1 M, pH 7.0 1R. W. Holley, Biochem. Biophys. Res. Commun. 10, 186 (1963). 2K. C. Smith, E. Cordes, and R. S. Schweet, Biochim. Biophys. Acta 33, 286 (1959).
[79]
SOLUBLE RNA
YEAST
603
SALTING OUT OF Y E A S T s-RNA F R O M W A T E R WITH AMMONIUM SULFATE. RNA-CONCENTRATION 5mg/ml --I
I00
I
I
I
I
90 ~
~-
I v I I . . . . . . . . . . ~..__~
I00
r"
o~ o ~
90
:
o -,..x° 7o
~.: ......
,/ . . ~ < ; " i: .......... 4.."~!..-,!.-A
80 __
p
oPER.AE \
sc
oo X / , %::/
E 5C O
' ~// /o ../~
t",J 4C
PRECIPITATE
W':/
~:.:,.--o
L:J: /
7o o
z
o,'.,'
6o x
z£" .i
/ ¢J
80:1. E
• VALtNE . . . . . . . . "TYROSINE. . . . . • BERINE ..........
o ALANiNE ........
50 ~> - -
40 (~
9t
20
20 % .
,o
• ,.,"
%
o
oZ.',.~ ..... 40
45
50
55
60
65
,o 70
75
80.
85
90
% AMMONIUM SULFATE SATURATION
FIG. 1. Salting out of sRNA with ammonium sulfate. Different amounts of ammonium sulfate were added to aliquots of an sRNA solution. After 20 hours at 4 °, the mixtures were centrifuged in the cold, and A ~ of the supernatants was measured. The precipitates were dissolved in buffer solution, and A~0 and amino acid acceptor activity were determined. Solid lines refer to A~0, dotted and dashed lines to total activity (A~o × amino acid acceptor activity, where acceptor activity of unfractionated sRNA is taken as 1.0). The precipitate formed at 45% ammonium sulfate saturation was recovered, dialyzed against distilled H:O, and subjected to a second complete salting-out experiment. The small but significant amount of valyl-acceptor RNA in this material, which is normally discarded, precipitated mainly between 55% and 70% ammonium sulfate saturation, as does the bulk of valyl-sRNA. This result indicates that nonspecific coprecipitation of sRNA, rather than a selective removal of certain sl%NA molecules, occurs at 45% ammonium sulfate saturation.
Phenol, freshly redistilled, water-saturated (at room temperature) Diethyl ether, A.R., (peroxide free), water-saturated Ammonium sulfate, A.R., pH of a 5% solution----5.2- 5.4 Dialysis tubing, Visking size 20 D.C. The tubing is immersed for 24 hours in 0.01 M Na~-EDTA, pH 7.0, and then boiled for 15 minutes in each of three changes of distilled water. Sephadex G-100 (Pharmacia Fine Chemicals, Inc.). The gel is added slowly under vigorous stirring to a large volume of 0.05 M potassium phosphate, pH 6.8, and boiled for 3 hours. After 10 minutes the gel has settled and the supernatant is decanted; hot distilled water (60-80 °) is added, and the gel is resuspended. The gel is washed with hot water four more times, then at room temperature several times with 1 M NaC1 + 0.01 M Tris-HC1 +
604
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[79]
0.001 M EDTA, pH ~--7.5. Immediately before packing, the gel suspension is transferred to a large suction flask and briefly deaerated under vacuum. Procedure. Five grams of crude sRNA is dissolved in 1000 ml of 0.2 M NaC1 ~ 0.01 M Tris pH ~ 7.5; 1500 ml of cold ethanol is slowly added to the solution with stirring, and the preparation is left overnight at 4 °. The supernatant is decanted and discarded; the precipitate is recovered by low speed centrifugation and washed once with 8 0 ~ ethanol, once with 95% ethanol, and dried in v a c u o over P205 at room temperature. The dry residue is dissolved in 200 ml of 0.001 M EDTA, pH ~ 7.0 and shaken vigorously with an equal volume of phenol for 10 minutes at room temperature. The emulsion is chilled and the phases are separated by low speed centrifugation in the cold. The top (aqueous) layer is removed by aspiration and saved. The remaining material is briefly extracted with 50 ml 0.001 M EDTA, and after low-speed centrifugation the small clear top layer is recovered by aspiration, taking care to avoid contamination with interphase material. The combined aqueous layers are briefly extracted four times with ether; residual ether in the aqueous phase is removed by bubbling filtered nitrogen through the solution. Solid ammonium sulfate is slowly added with stirring at room temperature in the proportion of 250 g to 1000 ml RNA solution (43~ saturation at 4°). The solution is then left at 4 ° for 20 hours, during which time a precipitate slowly develops. The precipitate is discarded after low speed centrifugation in the cold. More ammonium sulfate, 360 g to 1000 ml RNA solution (final concentration, 95% saturation at 4°), is added to the supernatant, and after 16-20 hours at 4 ° the precipitate is recovered by low speed centrifugation and suspended in 20-30 ml distilled H20. The sRNA is dialyzed, first against two changes of 50-100 volumes of 0.2 M NaC1 ~ 0.05 M EDTA, pH ~--7.0 at 4 ° over 24 hours, then against 3 changes of 1 M NaC1 for 48 hours, and finally against 5 changes of distilled H20 over 30 hours. Efficient dialysis is achieved by agitating the dialyzate. Extensive stretching of the dialysis sac can occur during the dialysis against H20, resulting in slight permeability of the membrane to sRNA2 The losses resulting thereby may be avoided by replacing the sac once or twice during this step. sRNA is recovered from the clear solution by freeze-drying. The white, fluffy solid is rapidly transferred to glass vials with air-tight 3j. Goldstein a n d L. C. Craig, J. A m . Chem. Sac. 82, 1833 (1960).
[79]
YEAST SOLUBLE RNA
605
screw caps and stored at room temperature or 4 °. The yield is 2.5-4.0 g, depending on the purity of the starting material. Alter~ative Procedure. After the initial ethanol precipitation and phenol treatment, the dry residue is dissolved in 80 ml of 1 M NaC1 Jr 0.01 M Tris-HC1-}- 0.001 M EDTA, pH ~7.5, heated at 40 ° for 30 minutes, and cooled to room temperature. The heating step releases some sRNA trapped as aggregates or in inactive conformations. If the preparation contains high-molecular weight RNA, a small precipitate can develop which should be removed by low-speed centrifugation. The sRNA solution is then applied to a column of Sephadex G-100 (5)< 160 cm), equilibrated with the same solvent at room temperature. The eluant is
400--
TyrAIo
300200 --
~ii
A258
S
100- Kd=O~/ 0 lOOO 1300I 700
Phe/
/
1 ~' 1600
1900
Kd=l$ T 22oo
EFFLUENT (ml)
Fro. 2. Sephadex G-100 gel filtration of crude.yeast sRNA. The fractions within the main peak were assayed for alanine-, phenylalanine-, serine-, tyrosine-, and valine-acceptor activities; the peak fraction for each activity is indicated. K~---distribution coefficient. passed through the column under a hydrostatic pressure of 50 cm. The effluent is collected in 15-20 ml fractions, and the ultraviolet absorption of each fraction is measured. A typical elution profile is shown in Fig. 2. The material appearing before the main peak consists of aggregated sRNA as well as ribosomal RNA (degraded high-molecular weight rRNA and 5 S rRNA). Some fractionation of the different amino acid-specific sRNA molecules occurs within the main peak; thus, the phenylalanineand serine-accepting molecules appear on the ascending slope. It is therefore important to recover the entire main peak if fractionation of the sRNA is to be avoided. The fractions within the peak are pooled, and the sRNA is precipitated and washed with ethanol as earlier. The precipitate is suspended in 20-30 ml distilled H20, and dialyzed and freeze-dried as above.
606
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[79]
Properties 4 The amino acid acceptor activity per milligram of the purified s R N A is 1.2-]..8 times t h a t of the starting material. The preparation has a phosphorus content of 9.0%. After exhaustive hydrolysis by pancreatic RNase, the only nucleosides observed (thin-layer chromatography) are cytidine and adenosine. The content of protein (biuret) is < 0 . 5 % , D N A < 0.1%, and starch (as iodine-reactive material) < 0 . 1 % . The product is free from nuclease activity and can be kept in solution at 20 ° for 48 hours without decrease in amino acid acceptor activity or molecular PROPERTIES OF YEAST s R N A a,b
Data obtained in
Parameter 22° AX% e(P) M~ (osmotic pressure) M~ (sedimentation equilibrium) M~ (sedimentation equilibrium) Partial specific volume (after dialysis against excess solvent) S~0.~
0.2 M NaC1 -{- 0.01 M phosphate (Na+) -{0.0005 M EDTA, pH 6.85
0.15 M KC1% 0.01 M cacodylate (K+) + 0.005 M MgC12 -t0.0005 M EDTA, pH 7.0
215 7400 26,500 _ 300 26,300 _+ 300
209 7200 ---
27,400 ± 600
--
0.531 ± 0.002 ml/g 4 . 0 0 ± 0 . 0 5 S ( a t 2 0 °)
0.505 ± 0.002 ml/g 4 . 1 0 ± 0 . 0 5 S ( a t 3 0 °)
a Values for sRNA largely devoid of terminal adenosine residue. b Ultraviolet optics have been used in experiments with the analytical ultracentrifuge. weight (if plastic gloves and carefully cleaned glassware have been used in the preparative work in order to minimize contamination with "finger nuclease"5). The w a t e r content of the freeze-dried material is 6-10%. After storage at room t e m p e r a t u r e for 6 months, no change was observed in biological activity or physical chemical solution properties. D r y i n g over P20s at room t e m p e r a t u r e reduces the w a t e r content to ~ 2 % without 4T. Lindahl, D. D. Henley, and J. R. Fresco, J. A m . Chem. Soc. 87, 4961 (1965); D. D. Henley, T. Lindahl, and J. R. Fresco, Proc. Natl. Acad. Sci. U.S. 55, 191 (lo66). ~R. W. Holley, J. Apgar, and S. H. Merrill, J. Biol. Chem. 236, PC42 (1961).
[80]
MAMMALIAN R~A
607
loss of biological activity. Solid RNA is very hygroscopic and should be kept protected from air. Some physical properties of the purified material are given in the table. Acknowledgment This work was supported by grant~ from the National Science Foundation (GB-492), the National Institutes of Health (GM-07654) and the American Heart Association.
[80] Preparation
of R N A
from Mammalian
Ribosomes
By Klvm MOLDAVE
Rat liver ribosomes, purified by several washes in solutions containing high concentrations of magnesium ions as described in this volume [56a], are extracted with a mixture of sodium dodecyl sulfate and watersaturated phenol. The RNA is recovered from the aqueous phase by precipitation with ethanol, and then the high molecular weight ribosomal RNA is resolved from low molecular weight sRNA by molecular sieve chromatography. 1 Reagents
2-Amino-2-hydroxymethylpropane-l,3-diol (Tris)-HC1 buffer, 0.01 M, pH 7.4 Sodium dodecyl sulfate, 3%, in 0.01 M Tris buffer, pH 7.4 Freshly prepared water-saturated phenol (at 4 ° ) Potassium acetate, 20%, adjusted to pH 5.0 with acetic acid Potassium acetate, 20%, pH 5.0 Extraction o] Nucleic Acids. Approximately 50 mg of purified ribosomes ~ are resuspended by gentle homogenization in 20 ml of 0.01 M Tris-HC1 buffer, pH 7.4. An equal volume of 3% sodium dodecyl sulfate is added, and the suspension is stirred with a magnetic mixer for 10 minutes at room temperature. Forty milliliters of water-saturated phenol is then added and the mixture is stirred vigorously for 1 hour at 4 °. All subsequent steps are carried out at 4 °. The resulting emulsion is centri-
1R. P. Sutter and K. Moldave, J. Biol. Chem. 241, 1698 (1966). 'E. Gasior and K. Moldave, J. Biol. Chem. 240, 3346 (1965).