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Alkaline hydrolysis of oligoribonucleotides on chromatographic paper
ANALYTICAL
54, 213-222 (1973)
BIOCHEMISTRY
Alkaline
Hydrolysis
of Oligoribonucleotides
on Chromatographic MICHAEL Institute
Paper1T2
W. KONRAD
of Molecular
University
Biology and Department oj Chemistry, of California, Los Angeles, California 90024
Received October 13, 1972; accepted January 9, 1973 A method is described which permits the alkaline hydrolysis of oligoribonucleotides while on chromatography paper. In one example of this technique, the dinucleotides from a pancreatic RNase digest are characterized. In the second example, some new information about the diversity of sequences found at the 5’-triphosphate termini of bacterial RNA is obtained. There are at least three different pancreatic RNase fragments beginning with adenosine, two with guanosine, and at least. three RNase Tl fragments beginning with adenosine.
Two-dimensional paper electrophoresis, or chromatography, or combinations of the two, is most often used to achieve the separation of a mixture of compounds for which a one-dimensional separation is not sufficient. If, however, the compounds are chemically or enzymatically treated after separation in the first dimension, development in the second can be used to reveal the effect of t’hat treatment. One example from the literature is the diagonal method of Hartley (1)) in which peptides linked by disulfide bonds are oxidized to cysteic acid peptides after development in the first direction and are then identified as the class with an altered mobility in the second, lying off the diagonal. Another is the diagonal method used by Dahlberg (2) to identify the oligonucleotide originating from the 3’ end of an RNA chain after RNase digestion. Since the 3’ terminal fragment is usually the only one not having a phosphate group which can be removed by alkaline phosphatase after electrophoresis in the first dimension, it will be found on the diagonal after electrophoresis in the second. In this communication, I describe two examples of the usefulness of alkaline hydrolysis of oligoribonucleotides after electrophoresis. The first ’ Reprints of this paper mill not be available within the United States. ’ Chemistry Department, University of California at Los Angeles, publication No. 3944. 213 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
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allows identification of some of the fragments resulting from the digestion of RNA by pancreatic ribonuclease. The second allows the specific visualization of ribonuclease fragments coming from the original 5’ end of bacterial RNA molecules and gives some new information about the diversity of nucleotide sequences found there. MATERIALS
AND
ME’THODS
Incorporation of Radioactive Phosphate into Bacteria. Escherichia c&i, strain K12 W3350 SR, maintained on egg slants (Difco Laboratories), was used to inoculate tryptone broth overnight cultures. A loo-fold dilution was made into M56 (3), a phosphate-buffered minimal medium 0.1% in glucose, and the culture again incubated at 32°C overnight. The cells were cent,rifuged and suspended in Tris medium, which consists of 0.1 M tris (hydroxymethyl) aminomethane-HCl (pH 7.3)) 0.01 M MgCl,, 0.001 M K,HPO,, 0.015 M (NH,)S04, 0.05 M KCl, 2 ,IJM FeS04; and the concentration adjusted to give a final turbidity, over a l-cm light path, at 660 nm, of 0.1. After growth to a turbidity of about 0.4 (typically 3.5 hr at 32”C), the cells were again centrifuged and resuspended in Tris medium, 0.2 mM phosphate. Long-term labeling of stable RNA was achieved by addition of 3ZP0,3- (International Chemical and Nuclear Corporation; supplied carrier-free in 0.02 M HCl, and adjusted to pH 7.5 with Tris base just before use) at a final concentration of 0.2 mCi/ml or less to a culture at a turbidity of 0.5 and allowing isotope incorporation to continue for at least one generation time. Short-term labeling of unstable RNA was accomplished by suspension of cells in the low-phosphate Tris medium at a turbidity of 2.5. In order to follow the depletion of phosphate in the medium, a very small amount of 32P0,3- (1 #&‘ml) was added, and 0.05-ml samples were taken every 5 min, diluted into 10 ml of cold 0.1 M NaCl, 0.01 M sodium azide in 0.1 M sodium phosphat’e buffer (pH 7), and filtered through 0.45-,prn pore size Millipore filters which were then washed with 10 ml of the same buffer. The amount of radioactivity in the filtrate, and thus the amount of phosphate left in the medium, was then measured as Cerenkov radiation (4) using a Packard liquid scintillation spectrometer. The decrease in phosphate was approximately linear during the time required to reach 0.05 mM, typically 20 min, when additional 32P043- was added to a final concentration of about 2 mCi/ml. Incorporation was terminated by dilution into 2 vol of cold 0.1 M phosphate, 0.01 M azide (pH 7). After centrifugation, the cells were washed in 0.10 M sodium phosphate, 0.01 M Tris-HCl (pH 7 I, and resuspended in 5 ml of 0.10 M sodium phosphat.e, 0.001 M EDTA (pH 7). Purification of RNA. Lysis was obtained by addition of sodium dodecyl
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sulfate to a final concentration of 1% and incubation for 5 min at 45°C. Protein was removed by addition of an equal volume of phenol (equilibrated with 0.2 M Tris-HCl and adjusted to a final pH of 7.2) and Bentonite (5) at a final concentration of 0.5 mg/ml. After vigorous agitation, the suspension was centrifuged, and the top aqueous layer was again extracted with phenol as before. The aqueous layer was dialyzed for 18 hr against 12 liters of 0.01 M Tris-HCl, 0.10 M sodium phosphate, (pH 7.5) ; then made 0.01 M in MgCl,, and DNase (Worthington Biochemical Corporation, electrophoretically purified) added to achieve a final concentration of 20 ,pg/ml. The solution was held at 37°C for 20 min and then extracted with phenol. The aqueous layer was layered onto a 2 X 35 cm column of Bio-Gel P-30 (Bio-Rad Laboratories) equilibrated with 0.05 M triethylammonium bicarbonate (pH 7) and eluted with the same buffer. Most of the radioactivity is excluded from the gel and emerges after the void volume. This material was pooled, evaporated to dryness at 45”C, and dissolved in water. Digestion with Ribonuclease. Digestion with pancreatic RNase A (Code RASE, Worthington Biochemical Corporation) was performed at 37°C for 30 min at an enzyme concentration of 0.1 mg/ml, the RNA concentration typically at 0.7 mg/ml, in 0.1 M triethylammonium bicarbonate buffer and 0.001 M EDTA (pH 7.4). Digestion with ribonuclease Tl (Worthington Biochemical Corporation) was accomplished at the same buffer and substrate concentration, but at an enzyme concentration of 7 pg/ml and an incubation at 37°C for 2 hr. In order to remove traces of cyclic phosphate esters resulting from incomplete nuclease digestion, formic acid was then added to all digests to a final concentration of lo%, and the RNA was held at 45°C for 1 hr, evaporated to dryness, dissolved in water, and subjected to electrophoresis. Digestion with LiOH. The paper containing the oligonucleotides was sprayed with 0.5 M LiOH so that it was thoroughly wet, but avoiding excess liquid which causes spreading and streaking during the hydrolysis. The paper was then sandwiched between two sheets of 1/b-in.-thick plate glass, with silicone-based stopcock grease applied at the edges of the sheets to prevent evaporation. Care must be taken to keep the grease at least 4 cm from the paper, or one risks it running between the plates and being absorbed by the paper. The weight of the plate glass is sufficient to provide an adequate seal, and clamps at the edges are not required. The glass should be as level as possible, or the compounds will drift across the paper, especially if excess LiOH has been used. Incubation was carried out in a heated cabinet, typically at 40°C for 16 hr. When looking for the tetraphosphates at the ends of RNA chains, com-
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FIG. 1. Electrophoretic distribution and identification of dinucleotides from a pancreatic RNase digest of bacterial RNA. (A) Autoradiograph made after electrophoresis in the second dimension. (B) Tracing showing the position of the four dinucleotides after electrophoresis in the first dimension with 7% formic acid (as rcdetermined by autoradiography) (. *.e.), the position of the mononucleotides sulting from alkaline hydrolysis of these dinucleotides and electrophoresis in a second dimension with 0.05 M sodium citrate (pH 4.0) (as determined by autoradiography) (-), and the position of the four 2’(3’) monophosphates applied in a mixture at the three positions marked with arrows before electrophoresis in the second dimension (as determined under a uv lamp) (- - -).
plete hydrolysis is most important, and incubation was usually carried out for 30 hr. Neutralization was accomplished after the paper had dried by spraying with 2.0 M acetic acid. After the paper had again dried, the lithium acetate was removed by washing in a 2 liter graduated cylinder filled with methyl alcohol (technical grade) at 4°C. Control experiments, in which [3H]ATP had been spotted on the paper before hydrolysis along with the oligonucleotides, revealed that more than 90% of the ATP could be recovered.
HYDROLYSIS
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SECOND DIMENSION
ORIG
I-‘, ,*-\ ‘,Cp: I BP I \, ‘,-,I
FIG.
c-.\ ,-\ ~,Gp;t,Up; \/ \,
1. (Continued).
Electrophoresis. Paper electrophoresis was performed in solvent-cooled tanks at approximately 25°C. Whatman chromatography paper, 3 MM and DEAE (DE 81, nominal capacity 3.5 pm-equivalents/cm2), were obtained from Reeve-Angel. RESULTS
Electrophoretic Distribution of Pancreatic RNase Dinucleotides. A fairly well-defined class of oligonucleotides can often be completely characterized if the members of the class are separated from each other, and the base composition of each species is then determined. The results of such a characterization are presented in Fig. 1. Approximately 20 000 cpm of 3ZP-labeled dinucleotides, purified from a pancreatic RNase digest of bacterial RNA (6) (labeled for several generations) was applied close to the corner of a 46 X 60 cm sheet of Whatman 3 MM chromatography paper. The paper was wet with 7% formic acid, and electrophoresis was performed in the first dimension for 70 min at 44 V/cm. After drying, the positions of the dinucleotides were determined by autoradiography with an exposure time of 6 hr. As can be seen in Fig. lB, the four pancreatic dinucleotides separated from each other, with the fastest-moving migrating 19 cm from the origin. The dinucleotides were then hydrolyzed with LiOH, and the products were subjected to electrophoresis in the second
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dimension in 0.05 M citrate (pH 4.0) for 3 hr at 30 V/cm. A mixture of the four 2’(3’) mononucleotides was applied to the three positions indicated by arrows, prior to electrophoresis in the second dimension, to serve as markers. Comparison of the final autoradiograph exposed for 18 hr (Fig. 1A) and the positions of the marker mononucleotides as visualized under uv irradiation demonstrates that the dinucleotides, in order of increasing electrophoretic mobility in the first dimension, are APCP, GPCP, APUP, and GPUP. Electrophoretic Distribution of RNase Fragments from the 5’ Termini of Bacterial RNA. RNA is polymerized in the 5’ to 3’ direction, with the 5’ end of the molecule retaining the three phosphates of the nucleotide triphosphate first incorporated into the chain (7-9). These three phosphates are released as part of a nucleoside tetraphosphate after alkaline hydrolysis, the fourth phosphate being the a phosphate of the second nucleotide incorporated int,o the RNA chain. This tetraphosphate is then a unique marker for the original 5’ end and distinguishes it from ends formed by nuclease action during maturation of st,able RNA, ends formed during messenger RNA breakdown, or ends formed during the purification of the RNA. The initial nucleotide in bacterial RNA is always a purine, and the two purine tetraphosphates resulting from alkaline hydrolysis, pppAp and pppGp, can be separated from each other as well as from the other products of hydrolysis by electrophoresis on DEAE paper in 0.05 M citrate (10) (pH 4.0). Using these facts, it is possible to learn something about the statistical distribution of nucleotide sequences at the 5’ ends of bacterial RNA. Cells were allowed to incorporate 32P0,3for 4.5 min; the RNA was extracted and digested with pancreatic RNase, applied near the corner of a 47 X 56 cm sheet of DEAE paper, which was then wet with 7% formic acid and electrophoresed for 18 hr at 20 V/cm. The oligonucleotides were then hydrolyzed with LiOH, and the products were electrophoresed in the second dimension for 4 hours at 50 V/cm in 0.05 M sodium citrate (pH 4.0). The autoradiograph of the resulting fingerprint is seen in Fig. 2. There are at least two distinct pppGp spots and three pppAp spots. A paper-strip scanner was used to quantitate the radioactivity in the tetraphosphate region, and it was found that the adenosine tetraphosphate spots contained 850, 720, and 920 cpm, while the guanosine spots contained 2400 and 4100 cpm, in order of decreasing mobility in the first dimension, out of a total of 2.1 X lo7 cpm applied to the paper at the origin. Since the electrophoretic mobility of oligonucleotides on DEAE paper in 7% formic acid is determined by both size and composition, with different species having similar mobilities, it is impossible to tell what the sequences of the terminal oligonucleotides were before hydrolysis with LiOH. It is possible, however, to conclude
HYDROLYSIS
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FIG. 2. Electrophoretic distribution of pancreatic RNase fragments from the 5’ triphosphate term% of bacterial RNA. A culture of exponentially growing bacteria was allowed to incorporate “P-phosphate for 4.5 min, and the RNA was purified and digested with pancreatic RNase as described in the methods section. 2.1 x 10’ cpm of the digest was applied to a 54 X 47 cm sheet of DEAE cellulose paper (lower right-hand corner in photograph) and electrophoresed for 18 hr in 7% formic acid at 30 V/cm (to the left). The paper was wet with 0.5 M LiOH, incubated. neutralized, the resulting salt removed, and electrophoresed 3 hr at 50 V/cm (upward in photography). Autoradiography for 6 days revealed the distribution of radioactivity.
that there is some diversity in initial sequences, in particular that the second nucleotide is not always a pyrimidine, for in that case, there would be only four terminal pancreatic RNase fragments (AC, AU, GC, GU) . One of the limits on resolution of terminal fragments using DEAE paper electrophoresis in the first dimension is due to the high charge on even the smaller ones, which causes them to migrate very slowly. To overcome this difficulty, a RNA digest (using Tl RNase in this experiment) was electrophoresed first on cellulose acetate at pH 3.5. In this system, the three phosphates at the 5’ end of the terminal fragments should increase their mobi1it.y relative to other oligonucleotides. The
220
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KONRAD
material was then transferred to one edge of a sheet of DEAE paper (11)) hydrolyzed as before with LiOH, and electrophoresed in the second dimension in 0.05 M sodium citrate (pH 4). No information about nucleotide sequences at the beginning of RNA chains starting with guanosine triphosphates is obtained, since the only product of Tl RNase digestion is pppGp. We can seen in Fig. 3, however, three distinct pppAp spots, thus indicating again that there is at least some heterogeneity of terminal nucleotide sequences at the 5’ ends of RNA chains, starting with adenosine. The spots were cut out, and the radioactivity measured with a
FIG. 3. Electrophoretic distribution of Tl RNase fragments from the 5’-triphosphate termini of bacterial RNA. Bacterial RNA labeled with “P-phosphate for 4.5 min was purified and digested with Tl RNase as described in the methods section. 3 X 10’ cpm of the digest, in 15 pl, was applied to a 70 X 5 cm strip of cellulose acetate, which was wet with a solution 5% in acetic acid and 0.5% in pyridine and electrophoresed for 90 min at SO V/cm. The oIigonucleotides were then transferred to the short edge of a 54 X 47 cm piece of DEAE paper, (the origin is to the right in photograph). Hydrolysis with LiOH was carried out as before, and the products electrophoresed in 0.05~ citrate buffer (pH 4) at 50 V/cm for 3 hr (upwards in photograph). Autoradiography for 10 days revealed the distribution of radioactivity.
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liquid scintillation counter, revealing 747, 1996, and 381 cpm in the adenosine tetraphosphate spots, in order of decreasing mobility in the first dimension, while the guanosine tetraphosphate spot contained 6360 cpm. The total applied at the origin had been 3 X lo7 cpm. DISCUSSION
I have described a relatively simple method of hydrolyzing oligoribonucleotides with alkali while on chromatography paper. This permits the simultaneous charact.erization of a large number of oligonucleotides by separating them with elect.rophoresis in one dimension and examination of the hydrolysis products after eleetrophoresis in the second dimension. The relatively simple problem of identifying the four dinucleotides produced by pancreatic RNasc, which is intended merely as an example here (their mobilities can be predicted in this system), could no doubt be accomplished almost as quickly by elution of each species,hydrolysis, and electrophoresis of the products. As the number of different species increases, however, the method described here should prove more efficient. Hydrolysis of ribonucleotides can be accomplished with volatile bases, thus eliminating the need for neutralization. Complete hydrolysis, however, even using concentrated ammonium hydroxide, requires 8 days at the moderate temperatures (45°C) needed to avoid deamination of some nucleotides (12). This technique is especially valuable as a solution to the second problem presented in this paper: the characterizat’ion of 5’-triphosphateterminated oligonucleotides. In a typical pulse-labeled bacterial RNA preparation, the radioactivity in these termini, or in RNase fragments containing these termini, is less than 1% of the total activity (9,lO). It is thus impossible to visualize them directly in the presence of the majority of oligonucleotides which come from the interior of RNA chains. Since they do represent such a small fraction of the total radioactivity, traces of products resulting from incomplete digestion can obscure them. Thus, while hydrolysis with a nonspecific nuclease might be an alternative in the experiment described in the preceding paragraph, it is unlikely to be here, since only comp1et.ehydrolysis gives satisfactory results (data not shown). Sequences at the triphosphate termini of RNAs have been studied by a number of others, usually using liquid chromatography with DE,4E cellulose or DEAE Sephadex in 7 M urea at pH 7 for the first fractionation of RNase fragment’s of the RNA. The terminal fragments have been identified in some cases by virtue of being selectively labeled using y-32P-triphosphates if the RNA was made in vitro (8,13), or by removing the phosphates from the RNA with phosphatase and replacing them with 32P using [r-““PI ATP as the donor and polynucleotide kinase (14).
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Neither of these techniques is suitable for the examination of RNA made in bacterial cells, since nucleotide triphosphates do not efficiently penetrate the cell wall to compete with those endogenously synthesized, and the vast majority of RNA molecules in the cell do not have three phosphates at the 5’ end. In other cases, the DEAE chromatographic fractions have been rechromatographed in a different system, or subjected to phosphatase digestion and rechromatography. The triphosphate-containing termini are separated from the internal fragments and can be sequenced (15). This technique can be very time-consuming, however, because the terminal fragments, having a larger charge to mass ratio than the fragments coming from the interior of the molecule, may chromatograph between two peaks of internal fragments. The method presented here has the advantage of detecting all RNA fragments containing 5’-triphosphate groups in a single two-dimensional fingerprint, yielding their mobility in the first dimension and the identity of the 5’-terminal nucleotide. Of course this ‘is far from a complete characterization. While such a technique is being developed, the alkaline digest fingerprint described here should be most helpful. ACKNOWLEDGMENTS The author is grateful to Winston Salser for pointing out the solubility of lithium salts in methanol and to Dohn Glitz and Donald Nierlich for helpful editing of this manuscript. The work was financially supported by a National Science Foundation research grant, GB-28127. REFERENCES 1. HARTLEY, B. S. (1970) Biochem. J. 119,805. 2. DAHLBERG, J. E. (1968) Nature (London) 220, 545. 3. WIESMMER, H., AND COHEN, M. (1960) Biochim. Biophys. Acta 3,417. 4. HABERER, H. K. (1965) Atomwirtschaft 10, 36. 5. FFLAENKEL-CONRAT, H., SINGER, B., AND TSUGITA, A. (1961) Virology 14, 54. 6. TOMLINSON, R. V., AND TENER, G. M. (1963) Biochemzktry 2, 697. 7. BREMER, H., KONRAD, M., GAINS, K., AND STENT, G. S. (1965) J. Mol. Biol. 13, 540. 8. MAITRA, U., AND HURWITZ, J. (1965) Proc. Nut. Acad. Sci. USA 54, 815. 9. JORGENSON, S., BUCH, L., AND NIERLICH, D. (1969) Science 164, 1067. 10. KONRAD, M., TOIVONEN, J.. AND NIERLICH, D. P. (1972) Nature New Biol. 238, 231. 11. SANDER, F., BROWNLEE, G. G., AND BAFEIELL, B. G. (1965) J. Mol. Biol. 13, 373. 12. BOULANGER, P., AND MONTREUIL, J. (1951) Bull. Sot. Chim. Biol. 33, 784. 13. SUGUIRA, M., ORAMOTO, T., AND TAKANAMI, M. (l(969) J. Mol. Biol. 43,299. 14. GLITZ, D. G. (1968) Biochemistry 7, 927. 15. DEWACHTER, R., VERHASSEL, J.-P., AND FIERS, W. (1968) Fed. Eur. Biochem. Sot. Letters 1, 93.
54, 213-222 (1973)
BIOCHEMISTRY
Alkaline
Hydrolysis
of Oligoribonucleotides
on Chromatographic MICHAEL Institute
Paper1T2
W. KONRAD
of Molecular
University
Biology and Department oj Chemistry, of California, Los Angeles, California 90024
Received October 13, 1972; accepted January 9, 1973 A method is described which permits the alkaline hydrolysis of oligoribonucleotides while on chromatography paper. In one example of this technique, the dinucleotides from a pancreatic RNase digest are characterized. In the second example, some new information about the diversity of sequences found at the 5’-triphosphate termini of bacterial RNA is obtained. There are at least three different pancreatic RNase fragments beginning with adenosine, two with guanosine, and at least. three RNase Tl fragments beginning with adenosine.
Two-dimensional paper electrophoresis, or chromatography, or combinations of the two, is most often used to achieve the separation of a mixture of compounds for which a one-dimensional separation is not sufficient. If, however, the compounds are chemically or enzymatically treated after separation in the first dimension, development in the second can be used to reveal the effect of t’hat treatment. One example from the literature is the diagonal method of Hartley (1)) in which peptides linked by disulfide bonds are oxidized to cysteic acid peptides after development in the first direction and are then identified as the class with an altered mobility in the second, lying off the diagonal. Another is the diagonal method used by Dahlberg (2) to identify the oligonucleotide originating from the 3’ end of an RNA chain after RNase digestion. Since the 3’ terminal fragment is usually the only one not having a phosphate group which can be removed by alkaline phosphatase after electrophoresis in the first dimension, it will be found on the diagonal after electrophoresis in the second. In this communication, I describe two examples of the usefulness of alkaline hydrolysis of oligoribonucleotides after electrophoresis. The first ’ Reprints of this paper mill not be available within the United States. ’ Chemistry Department, University of California at Los Angeles, publication No. 3944. 213 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
214
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W.
KONRAD
allows identification of some of the fragments resulting from the digestion of RNA by pancreatic ribonuclease. The second allows the specific visualization of ribonuclease fragments coming from the original 5’ end of bacterial RNA molecules and gives some new information about the diversity of nucleotide sequences found there. MATERIALS
AND
ME’THODS
Incorporation of Radioactive Phosphate into Bacteria. Escherichia c&i, strain K12 W3350 SR, maintained on egg slants (Difco Laboratories), was used to inoculate tryptone broth overnight cultures. A loo-fold dilution was made into M56 (3), a phosphate-buffered minimal medium 0.1% in glucose, and the culture again incubated at 32°C overnight. The cells were cent,rifuged and suspended in Tris medium, which consists of 0.1 M tris (hydroxymethyl) aminomethane-HCl (pH 7.3)) 0.01 M MgCl,, 0.001 M K,HPO,, 0.015 M (NH,)S04, 0.05 M KCl, 2 ,IJM FeS04; and the concentration adjusted to give a final turbidity, over a l-cm light path, at 660 nm, of 0.1. After growth to a turbidity of about 0.4 (typically 3.5 hr at 32”C), the cells were again centrifuged and resuspended in Tris medium, 0.2 mM phosphate. Long-term labeling of stable RNA was achieved by addition of 3ZP0,3- (International Chemical and Nuclear Corporation; supplied carrier-free in 0.02 M HCl, and adjusted to pH 7.5 with Tris base just before use) at a final concentration of 0.2 mCi/ml or less to a culture at a turbidity of 0.5 and allowing isotope incorporation to continue for at least one generation time. Short-term labeling of unstable RNA was accomplished by suspension of cells in the low-phosphate Tris medium at a turbidity of 2.5. In order to follow the depletion of phosphate in the medium, a very small amount of 32P0,3- (1 #&‘ml) was added, and 0.05-ml samples were taken every 5 min, diluted into 10 ml of cold 0.1 M NaCl, 0.01 M sodium azide in 0.1 M sodium phosphat’e buffer (pH 7), and filtered through 0.45-,prn pore size Millipore filters which were then washed with 10 ml of the same buffer. The amount of radioactivity in the filtrate, and thus the amount of phosphate left in the medium, was then measured as Cerenkov radiation (4) using a Packard liquid scintillation spectrometer. The decrease in phosphate was approximately linear during the time required to reach 0.05 mM, typically 20 min, when additional 32P043- was added to a final concentration of about 2 mCi/ml. Incorporation was terminated by dilution into 2 vol of cold 0.1 M phosphate, 0.01 M azide (pH 7). After centrifugation, the cells were washed in 0.10 M sodium phosphate, 0.01 M Tris-HCl (pH 7 I, and resuspended in 5 ml of 0.10 M sodium phosphat.e, 0.001 M EDTA (pH 7). Purification of RNA. Lysis was obtained by addition of sodium dodecyl
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sulfate to a final concentration of 1% and incubation for 5 min at 45°C. Protein was removed by addition of an equal volume of phenol (equilibrated with 0.2 M Tris-HCl and adjusted to a final pH of 7.2) and Bentonite (5) at a final concentration of 0.5 mg/ml. After vigorous agitation, the suspension was centrifuged, and the top aqueous layer was again extracted with phenol as before. The aqueous layer was dialyzed for 18 hr against 12 liters of 0.01 M Tris-HCl, 0.10 M sodium phosphate, (pH 7.5) ; then made 0.01 M in MgCl,, and DNase (Worthington Biochemical Corporation, electrophoretically purified) added to achieve a final concentration of 20 ,pg/ml. The solution was held at 37°C for 20 min and then extracted with phenol. The aqueous layer was layered onto a 2 X 35 cm column of Bio-Gel P-30 (Bio-Rad Laboratories) equilibrated with 0.05 M triethylammonium bicarbonate (pH 7) and eluted with the same buffer. Most of the radioactivity is excluded from the gel and emerges after the void volume. This material was pooled, evaporated to dryness at 45”C, and dissolved in water. Digestion with Ribonuclease. Digestion with pancreatic RNase A (Code RASE, Worthington Biochemical Corporation) was performed at 37°C for 30 min at an enzyme concentration of 0.1 mg/ml, the RNA concentration typically at 0.7 mg/ml, in 0.1 M triethylammonium bicarbonate buffer and 0.001 M EDTA (pH 7.4). Digestion with ribonuclease Tl (Worthington Biochemical Corporation) was accomplished at the same buffer and substrate concentration, but at an enzyme concentration of 7 pg/ml and an incubation at 37°C for 2 hr. In order to remove traces of cyclic phosphate esters resulting from incomplete nuclease digestion, formic acid was then added to all digests to a final concentration of lo%, and the RNA was held at 45°C for 1 hr, evaporated to dryness, dissolved in water, and subjected to electrophoresis. Digestion with LiOH. The paper containing the oligonucleotides was sprayed with 0.5 M LiOH so that it was thoroughly wet, but avoiding excess liquid which causes spreading and streaking during the hydrolysis. The paper was then sandwiched between two sheets of 1/b-in.-thick plate glass, with silicone-based stopcock grease applied at the edges of the sheets to prevent evaporation. Care must be taken to keep the grease at least 4 cm from the paper, or one risks it running between the plates and being absorbed by the paper. The weight of the plate glass is sufficient to provide an adequate seal, and clamps at the edges are not required. The glass should be as level as possible, or the compounds will drift across the paper, especially if excess LiOH has been used. Incubation was carried out in a heated cabinet, typically at 40°C for 16 hr. When looking for the tetraphosphates at the ends of RNA chains, com-
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FIG. 1. Electrophoretic distribution and identification of dinucleotides from a pancreatic RNase digest of bacterial RNA. (A) Autoradiograph made after electrophoresis in the second dimension. (B) Tracing showing the position of the four dinucleotides after electrophoresis in the first dimension with 7% formic acid (as rcdetermined by autoradiography) (. *.e.), the position of the mononucleotides sulting from alkaline hydrolysis of these dinucleotides and electrophoresis in a second dimension with 0.05 M sodium citrate (pH 4.0) (as determined by autoradiography) (-), and the position of the four 2’(3’) monophosphates applied in a mixture at the three positions marked with arrows before electrophoresis in the second dimension (as determined under a uv lamp) (- - -).
plete hydrolysis is most important, and incubation was usually carried out for 30 hr. Neutralization was accomplished after the paper had dried by spraying with 2.0 M acetic acid. After the paper had again dried, the lithium acetate was removed by washing in a 2 liter graduated cylinder filled with methyl alcohol (technical grade) at 4°C. Control experiments, in which [3H]ATP had been spotted on the paper before hydrolysis along with the oligonucleotides, revealed that more than 90% of the ATP could be recovered.
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SECOND DIMENSION
ORIG
I-‘, ,*-\ ‘,Cp: I BP I \, ‘,-,I
FIG.
c-.\ ,-\ ~,Gp;t,Up; \/ \,
1. (Continued).
Electrophoresis. Paper electrophoresis was performed in solvent-cooled tanks at approximately 25°C. Whatman chromatography paper, 3 MM and DEAE (DE 81, nominal capacity 3.5 pm-equivalents/cm2), were obtained from Reeve-Angel. RESULTS
Electrophoretic Distribution of Pancreatic RNase Dinucleotides. A fairly well-defined class of oligonucleotides can often be completely characterized if the members of the class are separated from each other, and the base composition of each species is then determined. The results of such a characterization are presented in Fig. 1. Approximately 20 000 cpm of 3ZP-labeled dinucleotides, purified from a pancreatic RNase digest of bacterial RNA (6) (labeled for several generations) was applied close to the corner of a 46 X 60 cm sheet of Whatman 3 MM chromatography paper. The paper was wet with 7% formic acid, and electrophoresis was performed in the first dimension for 70 min at 44 V/cm. After drying, the positions of the dinucleotides were determined by autoradiography with an exposure time of 6 hr. As can be seen in Fig. lB, the four pancreatic dinucleotides separated from each other, with the fastest-moving migrating 19 cm from the origin. The dinucleotides were then hydrolyzed with LiOH, and the products were subjected to electrophoresis in the second
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dimension in 0.05 M citrate (pH 4.0) for 3 hr at 30 V/cm. A mixture of the four 2’(3’) mononucleotides was applied to the three positions indicated by arrows, prior to electrophoresis in the second dimension, to serve as markers. Comparison of the final autoradiograph exposed for 18 hr (Fig. 1A) and the positions of the marker mononucleotides as visualized under uv irradiation demonstrates that the dinucleotides, in order of increasing electrophoretic mobility in the first dimension, are APCP, GPCP, APUP, and GPUP. Electrophoretic Distribution of RNase Fragments from the 5’ Termini of Bacterial RNA. RNA is polymerized in the 5’ to 3’ direction, with the 5’ end of the molecule retaining the three phosphates of the nucleotide triphosphate first incorporated into the chain (7-9). These three phosphates are released as part of a nucleoside tetraphosphate after alkaline hydrolysis, the fourth phosphate being the a phosphate of the second nucleotide incorporated int,o the RNA chain. This tetraphosphate is then a unique marker for the original 5’ end and distinguishes it from ends formed by nuclease action during maturation of st,able RNA, ends formed during messenger RNA breakdown, or ends formed during the purification of the RNA. The initial nucleotide in bacterial RNA is always a purine, and the two purine tetraphosphates resulting from alkaline hydrolysis, pppAp and pppGp, can be separated from each other as well as from the other products of hydrolysis by electrophoresis on DEAE paper in 0.05 M citrate (10) (pH 4.0). Using these facts, it is possible to learn something about the statistical distribution of nucleotide sequences at the 5’ ends of bacterial RNA. Cells were allowed to incorporate 32P0,3for 4.5 min; the RNA was extracted and digested with pancreatic RNase, applied near the corner of a 47 X 56 cm sheet of DEAE paper, which was then wet with 7% formic acid and electrophoresed for 18 hr at 20 V/cm. The oligonucleotides were then hydrolyzed with LiOH, and the products were electrophoresed in the second dimension for 4 hours at 50 V/cm in 0.05 M sodium citrate (pH 4.0). The autoradiograph of the resulting fingerprint is seen in Fig. 2. There are at least two distinct pppGp spots and three pppAp spots. A paper-strip scanner was used to quantitate the radioactivity in the tetraphosphate region, and it was found that the adenosine tetraphosphate spots contained 850, 720, and 920 cpm, while the guanosine spots contained 2400 and 4100 cpm, in order of decreasing mobility in the first dimension, out of a total of 2.1 X lo7 cpm applied to the paper at the origin. Since the electrophoretic mobility of oligonucleotides on DEAE paper in 7% formic acid is determined by both size and composition, with different species having similar mobilities, it is impossible to tell what the sequences of the terminal oligonucleotides were before hydrolysis with LiOH. It is possible, however, to conclude
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FIG. 2. Electrophoretic distribution of pancreatic RNase fragments from the 5’ triphosphate term% of bacterial RNA. A culture of exponentially growing bacteria was allowed to incorporate “P-phosphate for 4.5 min, and the RNA was purified and digested with pancreatic RNase as described in the methods section. 2.1 x 10’ cpm of the digest was applied to a 54 X 47 cm sheet of DEAE cellulose paper (lower right-hand corner in photograph) and electrophoresed for 18 hr in 7% formic acid at 30 V/cm (to the left). The paper was wet with 0.5 M LiOH, incubated. neutralized, the resulting salt removed, and electrophoresed 3 hr at 50 V/cm (upward in photography). Autoradiography for 6 days revealed the distribution of radioactivity.
that there is some diversity in initial sequences, in particular that the second nucleotide is not always a pyrimidine, for in that case, there would be only four terminal pancreatic RNase fragments (AC, AU, GC, GU) . One of the limits on resolution of terminal fragments using DEAE paper electrophoresis in the first dimension is due to the high charge on even the smaller ones, which causes them to migrate very slowly. To overcome this difficulty, a RNA digest (using Tl RNase in this experiment) was electrophoresed first on cellulose acetate at pH 3.5. In this system, the three phosphates at the 5’ end of the terminal fragments should increase their mobi1it.y relative to other oligonucleotides. The
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material was then transferred to one edge of a sheet of DEAE paper (11)) hydrolyzed as before with LiOH, and electrophoresed in the second dimension in 0.05 M sodium citrate (pH 4). No information about nucleotide sequences at the beginning of RNA chains starting with guanosine triphosphates is obtained, since the only product of Tl RNase digestion is pppGp. We can seen in Fig. 3, however, three distinct pppAp spots, thus indicating again that there is at least some heterogeneity of terminal nucleotide sequences at the 5’ ends of RNA chains, starting with adenosine. The spots were cut out, and the radioactivity measured with a
FIG. 3. Electrophoretic distribution of Tl RNase fragments from the 5’-triphosphate termini of bacterial RNA. Bacterial RNA labeled with “P-phosphate for 4.5 min was purified and digested with Tl RNase as described in the methods section. 3 X 10’ cpm of the digest, in 15 pl, was applied to a 70 X 5 cm strip of cellulose acetate, which was wet with a solution 5% in acetic acid and 0.5% in pyridine and electrophoresed for 90 min at SO V/cm. The oIigonucleotides were then transferred to the short edge of a 54 X 47 cm piece of DEAE paper, (the origin is to the right in photograph). Hydrolysis with LiOH was carried out as before, and the products electrophoresed in 0.05~ citrate buffer (pH 4) at 50 V/cm for 3 hr (upwards in photograph). Autoradiography for 10 days revealed the distribution of radioactivity.
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liquid scintillation counter, revealing 747, 1996, and 381 cpm in the adenosine tetraphosphate spots, in order of decreasing mobility in the first dimension, while the guanosine tetraphosphate spot contained 6360 cpm. The total applied at the origin had been 3 X lo7 cpm. DISCUSSION
I have described a relatively simple method of hydrolyzing oligoribonucleotides with alkali while on chromatography paper. This permits the simultaneous charact.erization of a large number of oligonucleotides by separating them with elect.rophoresis in one dimension and examination of the hydrolysis products after eleetrophoresis in the second dimension. The relatively simple problem of identifying the four dinucleotides produced by pancreatic RNasc, which is intended merely as an example here (their mobilities can be predicted in this system), could no doubt be accomplished almost as quickly by elution of each species,hydrolysis, and electrophoresis of the products. As the number of different species increases, however, the method described here should prove more efficient. Hydrolysis of ribonucleotides can be accomplished with volatile bases, thus eliminating the need for neutralization. Complete hydrolysis, however, even using concentrated ammonium hydroxide, requires 8 days at the moderate temperatures (45°C) needed to avoid deamination of some nucleotides (12). This technique is especially valuable as a solution to the second problem presented in this paper: the characterizat’ion of 5’-triphosphateterminated oligonucleotides. In a typical pulse-labeled bacterial RNA preparation, the radioactivity in these termini, or in RNase fragments containing these termini, is less than 1% of the total activity (9,lO). It is thus impossible to visualize them directly in the presence of the majority of oligonucleotides which come from the interior of RNA chains. Since they do represent such a small fraction of the total radioactivity, traces of products resulting from incomplete digestion can obscure them. Thus, while hydrolysis with a nonspecific nuclease might be an alternative in the experiment described in the preceding paragraph, it is unlikely to be here, since only comp1et.ehydrolysis gives satisfactory results (data not shown). Sequences at the triphosphate termini of RNAs have been studied by a number of others, usually using liquid chromatography with DE,4E cellulose or DEAE Sephadex in 7 M urea at pH 7 for the first fractionation of RNase fragment’s of the RNA. The terminal fragments have been identified in some cases by virtue of being selectively labeled using y-32P-triphosphates if the RNA was made in vitro (8,13), or by removing the phosphates from the RNA with phosphatase and replacing them with 32P using [r-““PI ATP as the donor and polynucleotide kinase (14).
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Neither of these techniques is suitable for the examination of RNA made in bacterial cells, since nucleotide triphosphates do not efficiently penetrate the cell wall to compete with those endogenously synthesized, and the vast majority of RNA molecules in the cell do not have three phosphates at the 5’ end. In other cases, the DEAE chromatographic fractions have been rechromatographed in a different system, or subjected to phosphatase digestion and rechromatography. The triphosphate-containing termini are separated from the internal fragments and can be sequenced (15). This technique can be very time-consuming, however, because the terminal fragments, having a larger charge to mass ratio than the fragments coming from the interior of the molecule, may chromatograph between two peaks of internal fragments. The method presented here has the advantage of detecting all RNA fragments containing 5’-triphosphate groups in a single two-dimensional fingerprint, yielding their mobility in the first dimension and the identity of the 5’-terminal nucleotide. Of course this ‘is far from a complete characterization. While such a technique is being developed, the alkaline digest fingerprint described here should be most helpful. ACKNOWLEDGMENTS The author is grateful to Winston Salser for pointing out the solubility of lithium salts in methanol and to Dohn Glitz and Donald Nierlich for helpful editing of this manuscript. The work was financially supported by a National Science Foundation research grant, GB-28127. REFERENCES 1. HARTLEY, B. S. (1970) Biochem. J. 119,805. 2. DAHLBERG, J. E. (1968) Nature (London) 220, 545. 3. WIESMMER, H., AND COHEN, M. (1960) Biochim. Biophys. Acta 3,417. 4. HABERER, H. K. (1965) Atomwirtschaft 10, 36. 5. FFLAENKEL-CONRAT, H., SINGER, B., AND TSUGITA, A. (1961) Virology 14, 54. 6. TOMLINSON, R. V., AND TENER, G. M. (1963) Biochemzktry 2, 697. 7. BREMER, H., KONRAD, M., GAINS, K., AND STENT, G. S. (1965) J. Mol. Biol. 13, 540. 8. MAITRA, U., AND HURWITZ, J. (1965) Proc. Nut. Acad. Sci. USA 54, 815. 9. JORGENSON, S., BUCH, L., AND NIERLICH, D. (1969) Science 164, 1067. 10. KONRAD, M., TOIVONEN, J.. AND NIERLICH, D. P. (1972) Nature New Biol. 238, 231. 11. SANDER, F., BROWNLEE, G. G., AND BAFEIELL, B. G. (1965) J. Mol. Biol. 13, 373. 12. BOULANGER, P., AND MONTREUIL, J. (1951) Bull. Sot. Chim. Biol. 33, 784. 13. SUGUIRA, M., ORAMOTO, T., AND TAKANAMI, M. (l(969) J. Mol. Biol. 43,299. 14. GLITZ, D. G. (1968) Biochemistry 7, 927. 15. DEWACHTER, R., VERHASSEL, J.-P., AND FIERS, W. (1968) Fed. Eur. Biochem. Sot. Letters 1, 93.