- Email: [email protected]
[86] Fractionation of RNA's by countercurrent distribution
644
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[85]
It has been found empirically that development of the chromatogram in less than 30 hours detectably decreases resolution, and times longer than 60 hours do not markedly improve the resolution. Small gradients (5-10 column volumes) and slow flows permit operation at high loading and elution of the RNA at concentrations of up to 3 mg/ml with no apparent loss of resolution from overloading. A column 1 meter long gives good resolution with initial loading of 50 mg RNA per square centimeter of the column bed. The techniques described here have been applied to samples ranging from less than 1 mg to 5 g. The procedures appear well suited for scale-up to even larger preparations. In some instances it is desirable to preload the sRNA with radioactive amino acids and chromatograph under conditions such that the alkali-labile amino acid ester remains attached to the sRNA. The DEAESephadex-urea column with a salt gradient at pH 4.5 and a complementary column, DEAE-cellulose at pH 4.2 in constant salt with a urea gradient are suitable for separation of aminoacyl-sRNA species (column types 4, 5, 6, and 8 of the table). The recovery of RNA from these columns is almost quantitative (95100%). The recovery of acceptor activities is believed to be not less than 70-80%. Fractions from DEAE-Sephadex-urea columns (pH 4.5) were stored in the refrigerator for several months without loss of acceptor activity. If the investigator is not attempting isolation of a certain pure sRNA but needs a single high resolution display of the pattern of acceptor activities, 5 a DEAE-cellulose column at pH 4.2 in 4 M urea developed with a sodium chloride gradient from 0.40 to 0.50 M (column type 6 of the table) exploits a maximum number of the chromatographic differences found among amino acid acceptor RNA's.
[86] F r a c t i o n a t i o n of R N A ' s b y C o u n t e r c u r r e n t D i s t r i b u t i o n
By B. P. DOCTOR Countercurrent distribution procedures have been employed successfully by several investigators for the separation and purification of transfer RNA's isolated from the yeast, Escherichia coli, and the rat liver. 1-21 Using these procedures it has been possible to fractionate grams L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Weissberger, ed.), 2nd ed., Vol. III, Pt. I, p. 149. Wiley (Interscience), New York, 1956. 2R. W. Holley and S. H. Merrill, J. Am. Chem. Soc. 81, 753 (1959). 3R. W. Holley, B. P. Doctor, S. H. Merrill, and F. M. Saad, Biochim. Biophys. Acta 35, 272 (1959).
[86]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
645
of transfer RNA's and obtain tens of milligrams of purified specific RNA. If one desires to separate various RNA's, it is possible to separate several RNA's specific for a single amino acid in a given species. These procedures are advantageous in that, using theoretical calculations, the extent of purity of a single homogeneous RNA can be determined. Finally, once the procedures are known, the method is quite a simple one to use. Three procedures that are commonly used for the separation and/or purification of specific transfer RNA will be presented here. Preparation of Transfer RNA's Suitable for Countercurrent Distribution In order to recover purified transfer RNA by eountercurrent distribution procedures, it is essential that no amino acid acceptor activity be lost during the process of purification. The major cause of loss of activity appears to be due to the "nucleases" present as contaminants in crude transfer RNA preparations. It is beyond the scope of this section to describe the procedure for the isolation of transfer RNA's. This is described elsewhere in this volume [76, 77]. It is thus essential that the starting material be tested for the presence of "nuclease" activity. This may be done by the procedures described by tIolley e t al. 22 If any ' R . W. Holley, J. Apgar, and B. P. Doctor, Ann. N.Y. Acad. Sci. 88, 745 (1960). 5B. P. Doctor, J. Apgar, and R. W. Holley, J. Biol. Chem. 256, 1117 (1961). eB. P. Doctor and C. M. Connelly, Biochim. Biophys. Res. Commun. 6, 201 (1961). 7H. G. Zachau, M. Tada, W. B. Lawson, and M. Schweiger, Biochim. Biophys. Acta 53, 221 (1961). 8j. Apgar, R. W. Holley, and S. It. Merrill, Biochim. Biophys. Acta 53, 220 (1961). ~J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). ~oM. Tada, M. Schweiger, and H. G. Zachau, Z. Physiol. Chem. 328, 85 (1962). 11H. Wiesmeyer, K. Kjellin, and H. G. Boman, Biochim. Biophys. Acta 61, 625
(1962). ~"J. Apgar ~.nd R. W. Holley, Bioehem. Biophys. Res. Commun. 8, 391 (1962). ~3B. Weisblum, S. Benzer, and R. W. Holley, Proc. Natl. Acad. Sci. U.8. 48, 1449
(1962). ~4B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3965 (1963). 15j. Goldstein, R. P. Bennett, and L. C. Craig, Proc. Natl. Acad. Sci. U~S. 51, 119 (1964). ~6j. Apgar and R. W. Holley, Biochem. Biophys. Res. Commun. 16, 121 (1964). 17G. W. Rushizky, H. A. Sober, C. M. Connelly, and B. P. Doctor, Bioehem. Biophys. Res. Commun. 18, 489 (1965). ~sW. Karau and H. G. Zachau, Biochim. Biophys. Acta 91, 549 (1964). ~oR. M. Hoskinson and H. G. Khorana, J. Biol. Chem. 240, 2129 (1965). ~oV. M. Ingram and J. A. SjSquist, Cold Spring Harbor Symp. Quant. Biol. 28, 133 (1963). 21G. yon Ehrenstein and D. Dais, Proc. Natl. Acad. Sci. U.S. 50, 81 (1963). R. W. ttolley, J. Apgar, and S. It. Merrill, J. Biol. Chem. 236, PC42 (1961).
646
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[86]
"nuelease" is found to be present in the RNA sample, it should be eliminated prior to use in countercurrent distribution. (The procedure to accomplish this is also given by Holley et al. 22) Procedure A - - P h o s p h a t e Buffer Solvent S y s t e m 23
All the procedures described here are designed for a 200-tube apparatus, each tube having the capacity to hold l0 ml of each phase. If an apparatus having different dimensions is employed, corresponding modifications according to these dimensions of the apparatus should be adopted. Increase or decrease of sample size proportionate to the volume of the solvent system does not appear to have appreciable effect on the pattern of distribution. However, this solvent system is quite sensitive to variations in temperature, apparently due to the change in composition of the solvent system. Increase in temperature appears to decrease the gross partition coefficient, whereas the reverse is true with a decrease in temperature. Thus, it is advisable to perform the distribution in constant temperature area (23 ° -+- 1°). Solvent System. Dipotassium hydrogen phosphate (550 g) and sodium dihydrogen phosphate monohydrate (850 g) are dissolved by adding 4000 ml glass distilled water (final volume 4470 ml). To this solution, 300 ml formamide and 1300 ml of isopropanol are added, and the contents are shaken vigorously several times to equilibrate the two phases. The twophase solvent system thus formed is allowed to equilibrate overnight at room temperature (23 ° + 1°). The ratio of volumes of upper and lower phase is approximately 1:1. Preparation o] RNA Sample and Procedures ]or Countercurrent Distribution. Approximately 2 g of yeast or rat liver transfer RNA or one g of E. coli transfer RNA is dissolved in 252 ml of phosphate buffer (described above). When all the RNA is dissolved, 17 ml formamide, 73.5 ml isopropanol, and 18 ml of lower phase are added and the contents are shaken to equilibrate the sample. The 200-tube countercurrent fractionator is charged with 10 ml each of previously separated upper and lower phases. Tube No. 200 is connected to tube No. 1 with a connecting tube which is filled with 10 ml of each phase. The conten~ of the first 20 tubes are emptied and replaced with 10 ml of each phase (previously separated and small amount of interface material discarded) containing RNA samples (approximately 18 tubes). The flask and separatory funnel used in the preparation of the sample are rinsed with enough upper and lower phase so as to fill the 19th and 20th tube with 10 ml of each phase. The machine is set for :~ R. W. Holley, J. Apgar, G. A. Everett, J. T. Madison, S. H. Merrill, and A. Zamir, Cold Spring Harbor Symp. Quant. Biol. 28, 117 (1963).
[86]
COUNTERCURRENT DISTRIBUTION--SOLUBLE R~A
647
200 transfers (2 minutes or 15 shakes and 4 minutes settling time), and the distribution is performed.
Recovery o] Fractions Suitable ]or Amino Acid Incorporation Assay. 2~-26 When the 200-transfer countereurrent distribution is completed, the machine is emptied and the contents of every five tubes across the distribution train are pooled so as to obtain 40 fractions. A small amount of upper and lower phase from each fraction is withdrawn and diluted to measure absorbance at 260 m#, corresponding amounts of fresh solvent system being used as a blank. The RNA content of each fraction is thus determined. The material is then transferred to a 500-ml separatory funnel and flask rinsed with a small amount of water and added to the sample. The sample is extracted with equal volumes of ether, and the lower phase is drained directly to another separatory funnel. To the lower phase is added 150 ml of water (thus adjusting the concentration of phosphate to approximately 0.4M), and an aqueous solution of cetyltrimethyl ammonium bromide (Cetavlon), and the contents were shaken gently. The amount of Cetavlon added should be approximately 5-6 times the amount of RNA present in the sample on weight/weight basis. After allowing to stand for 20-30 minutes, the contents were shaken. After 15 minutes, the quaternary complex of RNA floating at the interface is recovered by careful discarding of the lower phase and collection of the interface material in a small test tube. The sample is centrifuged, and the liquid is discarded. The RNA is then converted to the sodium salt by adding 0.5-1.0 ml of 2 M NaC1. Three volumes of 95% alcohol are added to precipitate the RNA. After the sample has stood in the cold to complete the precipitation, it is centrifuged and the precipitate is washed once with 80% and twice with 95% ethanol, and dried under vacuum. The recovery of A_~0m, absorbing material and amino acid acceptor activity is greater than 95% by this procedure. The RNA fractions thus obtained are dissolved completely or in part in distilled water and used for amino acid incorporation assays using several of the available procedures. For precise representation of the results, they can be described as follows: a plot is made showing the fraction number or tube number on the ordinate and milligrams or A~6om~ units per fraction of RNA on the left abscissa. Then from the amino acid incorporation assays, amino acid acceptor activity equivalent to starting RNA is calculated and expressed on the right abscissa. Such a curve clearly shows the extent of purification achieved by the counterA. D. Mirzabekov, A. T. Krutilina, V. I. Gorshkova, and A. A. Boev, 29, 1158 (1964). S H. G. Zachau, personal communication (1965). B. P. Doctor and J. E. Loebel, unpublished information (1965).
Biokhimiya
648
[86]
I S O L A T I O N AND F R A C T I O N A T I O N OF N U C L E I C ACIDS
current distribution for each R N A fraction. A representative result obtained for yeast transfer R N A by Apgar et al. 9 by such a procedure is shown in Fig. 1. 27
z
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Fro. 1. Two hundred-transfer countercurrent distribution of 500 mg of yeast "soluble RNA" in a solvent system similar to the one described under Procedure A. O-----Q, milligrams of RNA; A . . . . /x, alanine acceptor activity; O----C), valine acceptor activity; 0 . . . . Q, histidine acceptor activity; [] . . . . [-], tyrosine acceptor activity. Reproduced from J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). Procedure B - - - A m m o n i u m Sulfate Solvent System 6 The solvent system described here is a modification of the solvent described by K i r b y ? s The pattern of countercurrent distribution is essentially the same as the one obtained with the solvent system described in Procedure A. However, the solubility of R N A is greater in this solvent system t h a n in the phosphate buffer solvent system. Also, this solvent system is less sensitive to variations in temperature. S o l v e n t S y s t e m . Twelve hundred grams of a m m o n i u m sulfate was dissolved in approximately 3 liters of distilled water, and to it was added 40 ml of glacial acetic acid. After stirring thoroughly, 8 ml of concen:' The RNA fractions described in Figs. 1, 2, 4, 5, and 6 were isolated according to the procedures described in the original publications. The procedure described under "Procedure A" for the isolation of RNA fractions was developed recently in this laboratory. " K . S. Kirby, Biochim. Biophl/s. Acta 41, 338 (1960).
[85]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
649
trated ammonium hydroxide was added with continuous stirring. The solution was diluted to a total volume of 4 liters with distilled water (pH 4.0). To this, 160 ml of formamide and 1600 ml 2-ethoxyethanol were added and the contents were shaken vigorously. The two-phase solvent system thus formed was allowed to equilibrate overnight at room temperature ( 2 3 ° ± 1°). The ratio of volumes of upper to lower phase is approximately 1 • 1. Preparation o] Sample and Procedures for Countercurrent Distributian. Two grams of yeast or rat liver RNA or 1.0-1.5 g of E. coli RNA was dissolved in approximately 180 ml distilled water; 82.5 g ammonium sulfate, 2.75 ml glacial acetic acid, and 0.55 ml concentrated ammonium hydroxide were added gradually to the RNA sample with constant stirring. The solution was made up to a final volume of 275 ml. To this, 11 ml formamide and 110 ml 2-ethoxyethanol were added and the contents shaken. The procedures for the charging of the machine, introduction of sample, and performing the countercurrent distribution are the same as described for the phosphate buffer system. Recovery of RNA Fractions Suitable ]or Amino Acid Incorporation Assay. At the end of 200-transfer countercurrent distribution, the contents of every 5 tubes are pooled as described and extracted with equal volumes of ether. The lower phase is transferred to another separatory funnel and diluted with the addition of 350 ml water (the salt concentration being thus adjusted to approximately 0.4 M). Aqueous solution of cetyltrimethyl ammonium bromide was added and the contents gently shaken. The amount of Cetavlon should be approximately 5-6 times the amount of RNA present in the sample on weight/weight basis. The rest of the procedure is the same as described for the phosphate buffer system reported under Procedure A. Representative results obtained for E. coli transfer RNA by such a procedure are shown in Fig. 2. ~e Procedure C The solvent system described in this procedure was developed by Zaehau et al. ~,1° Some of the features of this system are as follows: (a) the tRNA is converted to the tri-n-butylammonium salt prior to countercurrent distribution; (b) the solvent system does not contain inorganic salts, which facilitates the recovery of the RNA after the distribution; (c) tRNA is very soluble in this solvent system (up to 200 mg yeast tRNA per milliliter of initial lower phase);is (d) the solvent system is less sensitive to temperature, but is sensitive to RNA concentration (partition coefficient increases slightly at higher concentration); and (e) for several tRNA's the distribution pattern is different from the patterns obtained with the systems described in procedures A and B.
650
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
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Fzo. 2. Two hundred-transfer countercurrent distribution of 300 mg of E. cell transfer RNA in a solvent system described under Procedure B. [] . . . . [-1, proline aceeptor activity; • . . . . O, lysine acceptor activity; (~). . . . Q, phenylalanine acceptor activity; A . . . . A, tyrosine acceptor activity; O--Q, Mg RNA per fraction. S o l v e n t S y s t e m . Two thousand milliliters n-butanol, 2600 ml distilled water, 200 ml tri-n-butylamine, 50 ml glacial acetic acid, and 440 ml peroxide-free n-butyl ether were mixed together and shaken. The ratio of the volumes of the two phases is approximately 1:1. Commercially available technical grade tri-n-butylamine should be purified prior to use in preparation of RNA sample and the solvent system. The procedure is as follows. 2~ The amine is heated with 15-20% (w/v) of phthalic acid anhydride until a homogeneous mixture is obtained. After cooling the amine is filtered or decanted, stirred for several hours with an equal volume of 2-3 N N a 0 H . The phases were allowed to separate, and the tri-n-butylamine phase was washed four times with water and vacuum distilled. With purified amine, ~ partition coefficient of 5 is obtained with 0.2 mg yeast t R N A tri-n-butylammonium salt per milliliter in a solvent system prepared with 20 parts n-butyl ether per 100 parts n-butanol; using technical grade amine, lower partition coefficient was obtained; however, this can be compensated by lowering the n-butyl ether content of the solvent system.
H. G. Zachau, Z. Physiol. Chem. 342, 98 (1965).
[8{)]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
651
Preparation o] RNA Sample and Procedures ]or Countercurrent Distribution. Two grams of tRNA to be distributed is dissolved in water, precipitated at 0 ° with HC1 to make the final concentration of the solution to 0.1 N HC1, centrifuged in the cold for 3 minutes at 5000 rpm, washed twice with 0.07 N HC1, and immediately dissolved by stirring with 30 ml water and 2-3 ml tri-n-butylamine. The solution is concentrated to 10-15 ml by flash evaporation and introduced into the first two tubes of the previously charged apparatus after equilibration with equal volumes of upper phase. Desired number of transfers are performed with 2 minutes of shaking and 10-15 minutes' settling time. The samples are pooled as described previously. The RNA from the fractions is recovered by precipitation with 2% potassium acetate and 1 volume of ethanol, washing with ethanol, and drying. The RNA fractions thus obtained are suitable for ultraviolet absorbance and amino acceptor activity determinations. Generally, over 95% of RNA and amino acid acceptor activity are recovered. A representative distribution pattern of yeast tRNA is shown in Fig. 3. 29 Recycling Very often it is desired to have a broader countercurrent distribution pattern than the available number of tubes of the apparatus permits. For example, several of the transfer RNA's specific for a single amino acid in a given species are either not separated or partially separated (overlapping peaks) in a 200-transfer countercurrent distribution. Thus, it is desirable t~ broaden the distribution pattern. This can be accomplished in two ways. (a) At the end of 200 transfers, the upper phase draining out of tube No. 200 is collected by means of a fraction collector, whereas fresh upper phase is continually fed into the apparatus through tube No. 1. This process can be continued until all the material has been removed from the distribution train. This procedure is better for broadening the distribution of the material in the first half of the apparatus (after 200 transfers). (b) In this procedure, several eountercurrent distributions are performed and a composite distribution pattern is obtained. For example, if 800 transfer countercurrent distribution of transfer RNA is desired, with a 200-tube apparatus, it can be performed as follows: A 200-transfer countercurrent distribution is performed by one of the three procedures described above. At the end of 200 transfers, the contents of tubes 51-200 are emptied and the tubes are refilled with fresh upper and lower phase (same amounts as starting distribution). The apparatus is set to recycle for 600 more transfers, thus performing a total of 800 transfers on the material contained in the first 50 tubes. Next, a second 200-transfer countereurrent distribution with exactly the same amount of RNA under
652
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[86]
mg RNA
20~ IO"
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the identical conditions is performed. This time, the contents of tubes 1-50 and 101-200 are emptied and replenished with fresh solvent. Additional 600 transfers are performed by recycling. Similarly, by performing two more countercurrent distributions with exactly the same amounts of RNA and recycling the material in tubes 101-150 and 151-200, respectively, for 800 transfers each, one can obtain a composite pattern of total of 800-transfer countercurrent distribution. An example of such a
[86]
COUNTERCURRENTDISTRIBUTION--SOLUBLE RNA
653
pattern for 400-transfer countercurrent distribution of yeast transfer RNA has been described2 Purification of R N A
In order that one may obtain a purified transfer RNA by countercurrent distribution, it is necessary to perform repeated distributions. Normally, this is not true for the separation of a mixture of a few compounds, however, transfer RNA constitutes a mixture of more than forty (probably as high as sixty-four) individual RNA's. The limitation of the number of available tubes in an apparatus, solubility of RNA in solvent system, the amount of effort involved in isolation of RNA fractions after the distribution is performed, changes in the solvent system during the long period required to perform a large number of transfers (in thousands), and, finally, the overall feasibility of such a venture indicates that repeated distribution to purify an RNA or two at a time is the preferable way to accomplish this goal. Several transfer RNA's (e.g., alanine, valine, histidine, tyrosine, 9 phenylalanine, 19 serine from the yeast 8,3° and tyrosine from E. coli 3~) have been purified by countercurrent distribution. It is preferable to perform initial countereurrent distribution in one solvent system, isolate the RNA fractions having the highest amino acid acceptor activity, and redistribute in another solvent system having a lower partition coefficient for the material to be redistributed. Procedure for the Purification of yeast alanine RNA ~4 will be described here. The method is presented here in such a manner that it could be extended for the purification of any other RNA's. A 200-transfer countercurrent distribution of 1 g of yeast transfer RNA's, using the procedure B as described above, was performed. At the end of 200 transfers, the contents of the apparatus were emptied, pooled, and extracted as described. Since the location of the alanine RNA peak in this distribution pattern is already established, one can save lots of effort by discarding the contents of tubes 51-200 and recovering the RNA from the fractions obtained from tubes 1-50. RNA fractions having the highest alanine acceptor activity (contents of tubes 16-35 in Fig. 4) were pooled and redistributed for 500 transfers (by the recycling procedure) in the following solvent system: 666 g dipotassium hydrogen phosphate and 1044 g of sodium dihydrogen phosphate monohydrate were dissolved in sufficient glass-distilled water to give a total volume of 6000 ml (1.9M, pH 6). To 5900 ml of this solution at room temperature, were added 590 ml of formamide and 2596 ml of isopropyl alcohol. The mixture was C. M. Connelly and B. P. Doctor, J. Biol. Chem. 241, 715 (1966). ~B. P. Doctor, J. E. Loebel, and M. Lipsitt, unpublished information (1965).
654
I S O L A T m N AND FRACTIONATION OF NUCLEIC ACIDS
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Fro. 4. Two hundred-transfer countercurrent distribution of approximately 1 g of yeast sRNA according to Procedure B. O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity; []-----r'-], tyrosine acceptor activity. Fractions marked "redistributed" were used in the distribution shown in Fig. 5. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963).
shaken vigorously. At the end of 500 transfers, the contents of the t u b e s were emptied and pooled, and R N A was isolated as described. The distribution pattern and alanine acceptor activity profile are shown in Fig. 5. The R N A fractions having the highest alanine acceptor activity were pooled and redistributed for 800 transfers in the same isopropyl alcohol-formamide-phosphate buffer system. The distribution pattern is shown in Fig. 6. At this point it is desirable to determine the extent of purity of R N A obtained by this method. The following criteria have been generally used: (1) One mole of R N A accepts one mole of amino acid. This criterion m a y not be rigidly applied since the enzymatic reaction of amino acid incorporation into the R N A molecule m a y not be completely charged. This in turn may be due to several factors, some of them are as follows: (a) presence of unlabeled amino acid in the enzyme preparation, (b) presence of trace
[86]
COUNTERCURRENT
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Fro. 5. Five hundred-transfer countercurrent distribution of alanine acceptor RNA fractions from two duplicate 200-transfer distributions such as shown in Fig. 4. This redistribution was performed in the solvent system described by J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). (Procedure A). O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963).
amounts of "nucleases" in the enzyme preparation, (c) deacylation of RNA during the removal process of unreacted amino acid, and (d) the contamination of other amino acids in labeled amino acid used. (2) Absorbance or weight of RNA, the amino acid acceptor activity and theoretically calculated curve 1 are in good agreement. (3) The specific amino acid aceeptor activity of RNA (mole RNA per mole amino acid incorporated) across the distribution pattern should be constant, and (4) no other amino acid is incorporated by the RNA. The results presented
656
ISOLATION
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Fz~. 6. Eight hundred-transfer countercurrent distribution of alanine acceptor RNA fraction derived as shown in Fig. 5. O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity; A . . . . /x, theoretical curve calculated for the partition coefficient of 0.1035 [L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Welssberger, ed.), 2nd ed., Vol. III, Pt. I, p. 149. Wiley (Interscience), New York, 1956]. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963). here are for the purpose of describing the method of purification of RNA. They do not rigidly fulfill all the criteria described above; however, they are in good agreement. It should be pointed out that it is possible to change the distribution pattern by making small changes in solvent system or performing the distribution at different temperature. For example, (1) by increasing or decreasing the formamide or isopropano] contents of phosphate buffer system, (2) by increasing or decreasing the formamide contents or by
[85]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
657
adding either 2-methoxyethanol or 2-butoxyethanol in an ammonium sulfate system, and (3) by increasing or decreasing n-butyl ether contents of tri-n-butylamine solvent system, one can obtain lower or higher partition coefficient of RNA. In addition to the separation and purification of transfer RNA's, countercurrent distribution procedures have been employed for the separation of other RNA's 32-3' and the separation of native DNA from the denatured DNA. 3~,~ These procedures have been employed also in the separation of oligonucleotides of the same chain lengths.37,3s Recently, we have extended our efforts in the separation of tri-, tetra-, and pentanucleotides obtained by pancreatic RNase digests of yeast high molecular weight RNA. 25 These experiments have given some indications as to the basis of separation by countercurrent distribution procedures. For example, in modified ammonium sulfate-containing solvent system, the oligonucleotides (of the same chain length) having higher guanylic acid content and lower adenylic acid content, have a lower partition coefficient than the oligonucleotides having higher adenylic acid content and lower guanylic acid content. This is one of several factors contributing to the separation of RNA's and oligonucleotides. Felsenfeld39 has recently shown that purified yeast serine transfer RNA appears to have a greater hydrogen bonded configuration than purified yeast alanine transfer RNA. From the countercurrent distribution pattern it can be observed that yeast alanine transfer RNA (low) and yeast serine transfer RNA (high) differ greatly in their partition coefficients. Thus, secondary and tertiary structures of RNA may be contributing to a great degree in the basis of separation of RNA's by countercurrent distribution. Acknowledgments Thanks are due to Dr. H. G. Zachau of the Institute of Genetics, University of Cologne, Germany; Dr. Jean Apgar, U. S. Nutrition Laboratory, Ithaca, New York; Dr. R. W. Holley, Cornell University, Ithaca, New York, and Mrs. Judith E. Loebel, of this division, for their valuable help in the preparation of this manuscript.
'*K. S. Kirby, Biochim. Biophys. Acta 41, 338 (1960). UT. Lif, G. Frieku, and P. A. Albertsson, J. Mol. Biol. 3, 727 (1961). *' K. S. Kirby, Biochim. Biophys. Acta 61, 506 (1962). ,5C. Kidson and K. S. Kirby, Biochim. Biophys. Acta 76, 624 (1963). ~*C. Kidson and K. S. Kirby, Biochim. Biophys. Acta 91, 627 (1964). G. J. McCormick and B. P. Doctor, Biochim. Biophys. Acta 76, 628 (1963). "B. P. Doctor and G. J. McCormick, Biochemistry 4, 49 (1965). G. Felsenfeld, personal communication (1965).
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[85]
It has been found empirically that development of the chromatogram in less than 30 hours detectably decreases resolution, and times longer than 60 hours do not markedly improve the resolution. Small gradients (5-10 column volumes) and slow flows permit operation at high loading and elution of the RNA at concentrations of up to 3 mg/ml with no apparent loss of resolution from overloading. A column 1 meter long gives good resolution with initial loading of 50 mg RNA per square centimeter of the column bed. The techniques described here have been applied to samples ranging from less than 1 mg to 5 g. The procedures appear well suited for scale-up to even larger preparations. In some instances it is desirable to preload the sRNA with radioactive amino acids and chromatograph under conditions such that the alkali-labile amino acid ester remains attached to the sRNA. The DEAESephadex-urea column with a salt gradient at pH 4.5 and a complementary column, DEAE-cellulose at pH 4.2 in constant salt with a urea gradient are suitable for separation of aminoacyl-sRNA species (column types 4, 5, 6, and 8 of the table). The recovery of RNA from these columns is almost quantitative (95100%). The recovery of acceptor activities is believed to be not less than 70-80%. Fractions from DEAE-Sephadex-urea columns (pH 4.5) were stored in the refrigerator for several months without loss of acceptor activity. If the investigator is not attempting isolation of a certain pure sRNA but needs a single high resolution display of the pattern of acceptor activities, 5 a DEAE-cellulose column at pH 4.2 in 4 M urea developed with a sodium chloride gradient from 0.40 to 0.50 M (column type 6 of the table) exploits a maximum number of the chromatographic differences found among amino acid acceptor RNA's.
[86] F r a c t i o n a t i o n of R N A ' s b y C o u n t e r c u r r e n t D i s t r i b u t i o n
By B. P. DOCTOR Countercurrent distribution procedures have been employed successfully by several investigators for the separation and purification of transfer RNA's isolated from the yeast, Escherichia coli, and the rat liver. 1-21 Using these procedures it has been possible to fractionate grams L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Weissberger, ed.), 2nd ed., Vol. III, Pt. I, p. 149. Wiley (Interscience), New York, 1956. 2R. W. Holley and S. H. Merrill, J. Am. Chem. Soc. 81, 753 (1959). 3R. W. Holley, B. P. Doctor, S. H. Merrill, and F. M. Saad, Biochim. Biophys. Acta 35, 272 (1959).
[86]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
645
of transfer RNA's and obtain tens of milligrams of purified specific RNA. If one desires to separate various RNA's, it is possible to separate several RNA's specific for a single amino acid in a given species. These procedures are advantageous in that, using theoretical calculations, the extent of purity of a single homogeneous RNA can be determined. Finally, once the procedures are known, the method is quite a simple one to use. Three procedures that are commonly used for the separation and/or purification of specific transfer RNA will be presented here. Preparation of Transfer RNA's Suitable for Countercurrent Distribution In order to recover purified transfer RNA by eountercurrent distribution procedures, it is essential that no amino acid acceptor activity be lost during the process of purification. The major cause of loss of activity appears to be due to the "nucleases" present as contaminants in crude transfer RNA preparations. It is beyond the scope of this section to describe the procedure for the isolation of transfer RNA's. This is described elsewhere in this volume [76, 77]. It is thus essential that the starting material be tested for the presence of "nuclease" activity. This may be done by the procedures described by tIolley e t al. 22 If any ' R . W. Holley, J. Apgar, and B. P. Doctor, Ann. N.Y. Acad. Sci. 88, 745 (1960). 5B. P. Doctor, J. Apgar, and R. W. Holley, J. Biol. Chem. 256, 1117 (1961). eB. P. Doctor and C. M. Connelly, Biochim. Biophys. Res. Commun. 6, 201 (1961). 7H. G. Zachau, M. Tada, W. B. Lawson, and M. Schweiger, Biochim. Biophys. Acta 53, 221 (1961). 8j. Apgar, R. W. Holley, and S. It. Merrill, Biochim. Biophys. Acta 53, 220 (1961). ~J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). ~oM. Tada, M. Schweiger, and H. G. Zachau, Z. Physiol. Chem. 328, 85 (1962). 11H. Wiesmeyer, K. Kjellin, and H. G. Boman, Biochim. Biophys. Acta 61, 625
(1962). ~"J. Apgar ~.nd R. W. Holley, Bioehem. Biophys. Res. Commun. 8, 391 (1962). ~3B. Weisblum, S. Benzer, and R. W. Holley, Proc. Natl. Acad. Sci. U.8. 48, 1449
(1962). ~4B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3965 (1963). 15j. Goldstein, R. P. Bennett, and L. C. Craig, Proc. Natl. Acad. Sci. U~S. 51, 119 (1964). ~6j. Apgar and R. W. Holley, Biochem. Biophys. Res. Commun. 16, 121 (1964). 17G. W. Rushizky, H. A. Sober, C. M. Connelly, and B. P. Doctor, Bioehem. Biophys. Res. Commun. 18, 489 (1965). ~sW. Karau and H. G. Zachau, Biochim. Biophys. Acta 91, 549 (1964). ~oR. M. Hoskinson and H. G. Khorana, J. Biol. Chem. 240, 2129 (1965). ~oV. M. Ingram and J. A. SjSquist, Cold Spring Harbor Symp. Quant. Biol. 28, 133 (1963). 21G. yon Ehrenstein and D. Dais, Proc. Natl. Acad. Sci. U.S. 50, 81 (1963). R. W. ttolley, J. Apgar, and S. It. Merrill, J. Biol. Chem. 236, PC42 (1961).
646
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[86]
"nuelease" is found to be present in the RNA sample, it should be eliminated prior to use in countercurrent distribution. (The procedure to accomplish this is also given by Holley et al. 22) Procedure A - - P h o s p h a t e Buffer Solvent S y s t e m 23
All the procedures described here are designed for a 200-tube apparatus, each tube having the capacity to hold l0 ml of each phase. If an apparatus having different dimensions is employed, corresponding modifications according to these dimensions of the apparatus should be adopted. Increase or decrease of sample size proportionate to the volume of the solvent system does not appear to have appreciable effect on the pattern of distribution. However, this solvent system is quite sensitive to variations in temperature, apparently due to the change in composition of the solvent system. Increase in temperature appears to decrease the gross partition coefficient, whereas the reverse is true with a decrease in temperature. Thus, it is advisable to perform the distribution in constant temperature area (23 ° -+- 1°). Solvent System. Dipotassium hydrogen phosphate (550 g) and sodium dihydrogen phosphate monohydrate (850 g) are dissolved by adding 4000 ml glass distilled water (final volume 4470 ml). To this solution, 300 ml formamide and 1300 ml of isopropanol are added, and the contents are shaken vigorously several times to equilibrate the two phases. The twophase solvent system thus formed is allowed to equilibrate overnight at room temperature (23 ° + 1°). The ratio of volumes of upper and lower phase is approximately 1:1. Preparation o] RNA Sample and Procedures ]or Countercurrent Distribution. Approximately 2 g of yeast or rat liver transfer RNA or one g of E. coli transfer RNA is dissolved in 252 ml of phosphate buffer (described above). When all the RNA is dissolved, 17 ml formamide, 73.5 ml isopropanol, and 18 ml of lower phase are added and the contents are shaken to equilibrate the sample. The 200-tube countercurrent fractionator is charged with 10 ml each of previously separated upper and lower phases. Tube No. 200 is connected to tube No. 1 with a connecting tube which is filled with 10 ml of each phase. The conten~ of the first 20 tubes are emptied and replaced with 10 ml of each phase (previously separated and small amount of interface material discarded) containing RNA samples (approximately 18 tubes). The flask and separatory funnel used in the preparation of the sample are rinsed with enough upper and lower phase so as to fill the 19th and 20th tube with 10 ml of each phase. The machine is set for :~ R. W. Holley, J. Apgar, G. A. Everett, J. T. Madison, S. H. Merrill, and A. Zamir, Cold Spring Harbor Symp. Quant. Biol. 28, 117 (1963).
[86]
COUNTERCURRENT DISTRIBUTION--SOLUBLE R~A
647
200 transfers (2 minutes or 15 shakes and 4 minutes settling time), and the distribution is performed.
Recovery o] Fractions Suitable ]or Amino Acid Incorporation Assay. 2~-26 When the 200-transfer countereurrent distribution is completed, the machine is emptied and the contents of every five tubes across the distribution train are pooled so as to obtain 40 fractions. A small amount of upper and lower phase from each fraction is withdrawn and diluted to measure absorbance at 260 m#, corresponding amounts of fresh solvent system being used as a blank. The RNA content of each fraction is thus determined. The material is then transferred to a 500-ml separatory funnel and flask rinsed with a small amount of water and added to the sample. The sample is extracted with equal volumes of ether, and the lower phase is drained directly to another separatory funnel. To the lower phase is added 150 ml of water (thus adjusting the concentration of phosphate to approximately 0.4M), and an aqueous solution of cetyltrimethyl ammonium bromide (Cetavlon), and the contents were shaken gently. The amount of Cetavlon added should be approximately 5-6 times the amount of RNA present in the sample on weight/weight basis. After allowing to stand for 20-30 minutes, the contents were shaken. After 15 minutes, the quaternary complex of RNA floating at the interface is recovered by careful discarding of the lower phase and collection of the interface material in a small test tube. The sample is centrifuged, and the liquid is discarded. The RNA is then converted to the sodium salt by adding 0.5-1.0 ml of 2 M NaC1. Three volumes of 95% alcohol are added to precipitate the RNA. After the sample has stood in the cold to complete the precipitation, it is centrifuged and the precipitate is washed once with 80% and twice with 95% ethanol, and dried under vacuum. The recovery of A_~0m, absorbing material and amino acid acceptor activity is greater than 95% by this procedure. The RNA fractions thus obtained are dissolved completely or in part in distilled water and used for amino acid incorporation assays using several of the available procedures. For precise representation of the results, they can be described as follows: a plot is made showing the fraction number or tube number on the ordinate and milligrams or A~6om~ units per fraction of RNA on the left abscissa. Then from the amino acid incorporation assays, amino acid acceptor activity equivalent to starting RNA is calculated and expressed on the right abscissa. Such a curve clearly shows the extent of purification achieved by the counterA. D. Mirzabekov, A. T. Krutilina, V. I. Gorshkova, and A. A. Boev, 29, 1158 (1964). S H. G. Zachau, personal communication (1965). B. P. Doctor and J. E. Loebel, unpublished information (1965).
Biokhimiya
648
[86]
I S O L A T I O N AND F R A C T I O N A T I O N OF N U C L E I C ACIDS
current distribution for each R N A fraction. A representative result obtained for yeast transfer R N A by Apgar et al. 9 by such a procedure is shown in Fig. 1. 27
z
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Fro. 1. Two hundred-transfer countercurrent distribution of 500 mg of yeast "soluble RNA" in a solvent system similar to the one described under Procedure A. O-----Q, milligrams of RNA; A . . . . /x, alanine acceptor activity; O----C), valine acceptor activity; 0 . . . . Q, histidine acceptor activity; [] . . . . [-], tyrosine acceptor activity. Reproduced from J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). Procedure B - - - A m m o n i u m Sulfate Solvent System 6 The solvent system described here is a modification of the solvent described by K i r b y ? s The pattern of countercurrent distribution is essentially the same as the one obtained with the solvent system described in Procedure A. However, the solubility of R N A is greater in this solvent system t h a n in the phosphate buffer solvent system. Also, this solvent system is less sensitive to variations in temperature. S o l v e n t S y s t e m . Twelve hundred grams of a m m o n i u m sulfate was dissolved in approximately 3 liters of distilled water, and to it was added 40 ml of glacial acetic acid. After stirring thoroughly, 8 ml of concen:' The RNA fractions described in Figs. 1, 2, 4, 5, and 6 were isolated according to the procedures described in the original publications. The procedure described under "Procedure A" for the isolation of RNA fractions was developed recently in this laboratory. " K . S. Kirby, Biochim. Biophl/s. Acta 41, 338 (1960).
[85]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
649
trated ammonium hydroxide was added with continuous stirring. The solution was diluted to a total volume of 4 liters with distilled water (pH 4.0). To this, 160 ml of formamide and 1600 ml 2-ethoxyethanol were added and the contents were shaken vigorously. The two-phase solvent system thus formed was allowed to equilibrate overnight at room temperature ( 2 3 ° ± 1°). The ratio of volumes of upper to lower phase is approximately 1 • 1. Preparation o] Sample and Procedures for Countercurrent Distributian. Two grams of yeast or rat liver RNA or 1.0-1.5 g of E. coli RNA was dissolved in approximately 180 ml distilled water; 82.5 g ammonium sulfate, 2.75 ml glacial acetic acid, and 0.55 ml concentrated ammonium hydroxide were added gradually to the RNA sample with constant stirring. The solution was made up to a final volume of 275 ml. To this, 11 ml formamide and 110 ml 2-ethoxyethanol were added and the contents shaken. The procedures for the charging of the machine, introduction of sample, and performing the countercurrent distribution are the same as described for the phosphate buffer system. Recovery of RNA Fractions Suitable ]or Amino Acid Incorporation Assay. At the end of 200-transfer countercurrent distribution, the contents of every 5 tubes are pooled as described and extracted with equal volumes of ether. The lower phase is transferred to another separatory funnel and diluted with the addition of 350 ml water (the salt concentration being thus adjusted to approximately 0.4 M). Aqueous solution of cetyltrimethyl ammonium bromide was added and the contents gently shaken. The amount of Cetavlon should be approximately 5-6 times the amount of RNA present in the sample on weight/weight basis. The rest of the procedure is the same as described for the phosphate buffer system reported under Procedure A. Representative results obtained for E. coli transfer RNA by such a procedure are shown in Fig. 2. ~e Procedure C The solvent system described in this procedure was developed by Zaehau et al. ~,1° Some of the features of this system are as follows: (a) the tRNA is converted to the tri-n-butylammonium salt prior to countercurrent distribution; (b) the solvent system does not contain inorganic salts, which facilitates the recovery of the RNA after the distribution; (c) tRNA is very soluble in this solvent system (up to 200 mg yeast tRNA per milliliter of initial lower phase);is (d) the solvent system is less sensitive to temperature, but is sensitive to RNA concentration (partition coefficient increases slightly at higher concentration); and (e) for several tRNA's the distribution pattern is different from the patterns obtained with the systems described in procedures A and B.
650
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
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Fzo. 2. Two hundred-transfer countercurrent distribution of 300 mg of E. cell transfer RNA in a solvent system described under Procedure B. [] . . . . [-1, proline aceeptor activity; • . . . . O, lysine acceptor activity; (~). . . . Q, phenylalanine acceptor activity; A . . . . A, tyrosine acceptor activity; O--Q, Mg RNA per fraction. S o l v e n t S y s t e m . Two thousand milliliters n-butanol, 2600 ml distilled water, 200 ml tri-n-butylamine, 50 ml glacial acetic acid, and 440 ml peroxide-free n-butyl ether were mixed together and shaken. The ratio of the volumes of the two phases is approximately 1:1. Commercially available technical grade tri-n-butylamine should be purified prior to use in preparation of RNA sample and the solvent system. The procedure is as follows. 2~ The amine is heated with 15-20% (w/v) of phthalic acid anhydride until a homogeneous mixture is obtained. After cooling the amine is filtered or decanted, stirred for several hours with an equal volume of 2-3 N N a 0 H . The phases were allowed to separate, and the tri-n-butylamine phase was washed four times with water and vacuum distilled. With purified amine, ~ partition coefficient of 5 is obtained with 0.2 mg yeast t R N A tri-n-butylammonium salt per milliliter in a solvent system prepared with 20 parts n-butyl ether per 100 parts n-butanol; using technical grade amine, lower partition coefficient was obtained; however, this can be compensated by lowering the n-butyl ether content of the solvent system.
H. G. Zachau, Z. Physiol. Chem. 342, 98 (1965).
[8{)]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
651
Preparation o] RNA Sample and Procedures ]or Countercurrent Distribution. Two grams of tRNA to be distributed is dissolved in water, precipitated at 0 ° with HC1 to make the final concentration of the solution to 0.1 N HC1, centrifuged in the cold for 3 minutes at 5000 rpm, washed twice with 0.07 N HC1, and immediately dissolved by stirring with 30 ml water and 2-3 ml tri-n-butylamine. The solution is concentrated to 10-15 ml by flash evaporation and introduced into the first two tubes of the previously charged apparatus after equilibration with equal volumes of upper phase. Desired number of transfers are performed with 2 minutes of shaking and 10-15 minutes' settling time. The samples are pooled as described previously. The RNA from the fractions is recovered by precipitation with 2% potassium acetate and 1 volume of ethanol, washing with ethanol, and drying. The RNA fractions thus obtained are suitable for ultraviolet absorbance and amino acceptor activity determinations. Generally, over 95% of RNA and amino acid acceptor activity are recovered. A representative distribution pattern of yeast tRNA is shown in Fig. 3. 29 Recycling Very often it is desired to have a broader countercurrent distribution pattern than the available number of tubes of the apparatus permits. For example, several of the transfer RNA's specific for a single amino acid in a given species are either not separated or partially separated (overlapping peaks) in a 200-transfer countercurrent distribution. Thus, it is desirable t~ broaden the distribution pattern. This can be accomplished in two ways. (a) At the end of 200 transfers, the upper phase draining out of tube No. 200 is collected by means of a fraction collector, whereas fresh upper phase is continually fed into the apparatus through tube No. 1. This process can be continued until all the material has been removed from the distribution train. This procedure is better for broadening the distribution of the material in the first half of the apparatus (after 200 transfers). (b) In this procedure, several eountercurrent distributions are performed and a composite distribution pattern is obtained. For example, if 800 transfer countercurrent distribution of transfer RNA is desired, with a 200-tube apparatus, it can be performed as follows: A 200-transfer countercurrent distribution is performed by one of the three procedures described above. At the end of 200 transfers, the contents of tubes 51-200 are emptied and the tubes are refilled with fresh upper and lower phase (same amounts as starting distribution). The apparatus is set to recycle for 600 more transfers, thus performing a total of 800 transfers on the material contained in the first 50 tubes. Next, a second 200-transfer countereurrent distribution with exactly the same amount of RNA under
652
ISOLATION AND FRACTIONATION OF NUCLEIC ACIDS
[86]
mg RNA
20~ IO"
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the identical conditions is performed. This time, the contents of tubes 1-50 and 101-200 are emptied and replenished with fresh solvent. Additional 600 transfers are performed by recycling. Similarly, by performing two more countercurrent distributions with exactly the same amounts of RNA and recycling the material in tubes 101-150 and 151-200, respectively, for 800 transfers each, one can obtain a composite pattern of total of 800-transfer countercurrent distribution. An example of such a
[86]
COUNTERCURRENTDISTRIBUTION--SOLUBLE RNA
653
pattern for 400-transfer countercurrent distribution of yeast transfer RNA has been described2 Purification of R N A
In order that one may obtain a purified transfer RNA by countercurrent distribution, it is necessary to perform repeated distributions. Normally, this is not true for the separation of a mixture of a few compounds, however, transfer RNA constitutes a mixture of more than forty (probably as high as sixty-four) individual RNA's. The limitation of the number of available tubes in an apparatus, solubility of RNA in solvent system, the amount of effort involved in isolation of RNA fractions after the distribution is performed, changes in the solvent system during the long period required to perform a large number of transfers (in thousands), and, finally, the overall feasibility of such a venture indicates that repeated distribution to purify an RNA or two at a time is the preferable way to accomplish this goal. Several transfer RNA's (e.g., alanine, valine, histidine, tyrosine, 9 phenylalanine, 19 serine from the yeast 8,3° and tyrosine from E. coli 3~) have been purified by countercurrent distribution. It is preferable to perform initial countereurrent distribution in one solvent system, isolate the RNA fractions having the highest amino acid acceptor activity, and redistribute in another solvent system having a lower partition coefficient for the material to be redistributed. Procedure for the Purification of yeast alanine RNA ~4 will be described here. The method is presented here in such a manner that it could be extended for the purification of any other RNA's. A 200-transfer countercurrent distribution of 1 g of yeast transfer RNA's, using the procedure B as described above, was performed. At the end of 200 transfers, the contents of the apparatus were emptied, pooled, and extracted as described. Since the location of the alanine RNA peak in this distribution pattern is already established, one can save lots of effort by discarding the contents of tubes 51-200 and recovering the RNA from the fractions obtained from tubes 1-50. RNA fractions having the highest alanine acceptor activity (contents of tubes 16-35 in Fig. 4) were pooled and redistributed for 500 transfers (by the recycling procedure) in the following solvent system: 666 g dipotassium hydrogen phosphate and 1044 g of sodium dihydrogen phosphate monohydrate were dissolved in sufficient glass-distilled water to give a total volume of 6000 ml (1.9M, pH 6). To 5900 ml of this solution at room temperature, were added 590 ml of formamide and 2596 ml of isopropyl alcohol. The mixture was C. M. Connelly and B. P. Doctor, J. Biol. Chem. 241, 715 (1966). ~B. P. Doctor, J. E. Loebel, and M. Lipsitt, unpublished information (1965).
654
I S O L A T m N AND FRACTIONATION OF NUCLEIC ACIDS
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Fro. 4. Two hundred-transfer countercurrent distribution of approximately 1 g of yeast sRNA according to Procedure B. O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity; []-----r'-], tyrosine acceptor activity. Fractions marked "redistributed" were used in the distribution shown in Fig. 5. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963).
shaken vigorously. At the end of 500 transfers, the contents of the t u b e s were emptied and pooled, and R N A was isolated as described. The distribution pattern and alanine acceptor activity profile are shown in Fig. 5. The R N A fractions having the highest alanine acceptor activity were pooled and redistributed for 800 transfers in the same isopropyl alcohol-formamide-phosphate buffer system. The distribution pattern is shown in Fig. 6. At this point it is desirable to determine the extent of purity of R N A obtained by this method. The following criteria have been generally used: (1) One mole of R N A accepts one mole of amino acid. This criterion m a y not be rigidly applied since the enzymatic reaction of amino acid incorporation into the R N A molecule m a y not be completely charged. This in turn may be due to several factors, some of them are as follows: (a) presence of unlabeled amino acid in the enzyme preparation, (b) presence of trace
[86]
COUNTERCURRENT
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Fro. 5. Five hundred-transfer countercurrent distribution of alanine acceptor RNA fractions from two duplicate 200-transfer distributions such as shown in Fig. 4. This redistribution was performed in the solvent system described by J. Apgar, R. W. Holley, and S. H. Merrill, J. Biol. Chem. 237, 796 (1962). (Procedure A). O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963).
amounts of "nucleases" in the enzyme preparation, (c) deacylation of RNA during the removal process of unreacted amino acid, and (d) the contamination of other amino acids in labeled amino acid used. (2) Absorbance or weight of RNA, the amino acid acceptor activity and theoretically calculated curve 1 are in good agreement. (3) The specific amino acid aceeptor activity of RNA (mole RNA per mole amino acid incorporated) across the distribution pattern should be constant, and (4) no other amino acid is incorporated by the RNA. The results presented
656
ISOLATION
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[86]
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150
NUMBER
Fz~. 6. Eight hundred-transfer countercurrent distribution of alanine acceptor RNA fraction derived as shown in Fig. 5. O O, milligrams of RNA per fraction; X . . . . X, alanine acceptor activity; A . . . . /x, theoretical curve calculated for the partition coefficient of 0.1035 [L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Welssberger, ed.), 2nd ed., Vol. III, Pt. I, p. 149. Wiley (Interscience), New York, 1956]. Reproduced from B. P. Doctor, C. M. Connelly, G. W. Rushizky, and H. A. Sober, J. Biol. Chem. 238, 3985 (1963). here are for the purpose of describing the method of purification of RNA. They do not rigidly fulfill all the criteria described above; however, they are in good agreement. It should be pointed out that it is possible to change the distribution pattern by making small changes in solvent system or performing the distribution at different temperature. For example, (1) by increasing or decreasing the formamide or isopropano] contents of phosphate buffer system, (2) by increasing or decreasing the formamide contents or by
[85]
COUNTERCURRENT DISTRIBUTION--SOLUBLE RNA
657
adding either 2-methoxyethanol or 2-butoxyethanol in an ammonium sulfate system, and (3) by increasing or decreasing n-butyl ether contents of tri-n-butylamine solvent system, one can obtain lower or higher partition coefficient of RNA. In addition to the separation and purification of transfer RNA's, countercurrent distribution procedures have been employed for the separation of other RNA's 32-3' and the separation of native DNA from the denatured DNA. 3~,~ These procedures have been employed also in the separation of oligonucleotides of the same chain lengths.37,3s Recently, we have extended our efforts in the separation of tri-, tetra-, and pentanucleotides obtained by pancreatic RNase digests of yeast high molecular weight RNA. 25 These experiments have given some indications as to the basis of separation by countercurrent distribution procedures. For example, in modified ammonium sulfate-containing solvent system, the oligonucleotides (of the same chain length) having higher guanylic acid content and lower adenylic acid content, have a lower partition coefficient than the oligonucleotides having higher adenylic acid content and lower guanylic acid content. This is one of several factors contributing to the separation of RNA's and oligonucleotides. Felsenfeld39 has recently shown that purified yeast serine transfer RNA appears to have a greater hydrogen bonded configuration than purified yeast alanine transfer RNA. From the countercurrent distribution pattern it can be observed that yeast alanine transfer RNA (low) and yeast serine transfer RNA (high) differ greatly in their partition coefficients. Thus, secondary and tertiary structures of RNA may be contributing to a great degree in the basis of separation of RNA's by countercurrent distribution. Acknowledgments Thanks are due to Dr. H. G. Zachau of the Institute of Genetics, University of Cologne, Germany; Dr. Jean Apgar, U. S. Nutrition Laboratory, Ithaca, New York; Dr. R. W. Holley, Cornell University, Ithaca, New York, and Mrs. Judith E. Loebel, of this division, for their valuable help in the preparation of this manuscript.
'*K. S. Kirby, Biochim. Biophys. Acta 41, 338 (1960). UT. Lif, G. Frieku, and P. A. Albertsson, J. Mol. Biol. 3, 727 (1961). *' K. S. Kirby, Biochim. Biophys. Acta 61, 506 (1962). ,5C. Kidson and K. S. Kirby, Biochim. Biophys. Acta 76, 624 (1963). ~*C. Kidson and K. S. Kirby, Biochim. Biophys. Acta 91, 627 (1964). G. J. McCormick and B. P. Doctor, Biochim. Biophys. Acta 76, 628 (1963). "B. P. Doctor and G. J. McCormick, Biochemistry 4, 49 (1965). G. Felsenfeld, personal communication (1965).