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Enzymatic synthesis of uniformly 32P-labeled polyribonucleotides and high-specific-activity ribonucleoside 5′-[α-32P]diphosphates
ANALYTICAL
BIOCHEMISTRY
144,291-295
(1985)
Enzymatic Synthesis of Uniformly 32P-Labeled Polyribonucleotides High-Specific-Activity Ribonucleoside 5’-[a-32P]diphosphates’ R. CANTOR
CHANGWONKANGANDCHARLES Department
and
of Human Genetics and Development, College of Physicians Columbia University, New York, New York 10032
and Surgeons,
Received June 2 1, 1984 Uniformly “P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5’-[ru-32P]ttiphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition. 0 1985 Academic press, hc. KEY WORDS: nucleic acid chemistry; myosin ATPase; polynucleotide phosphorylase; poly(U); poly(A); nucleoside diphosphates.
Synthetic oligo- and polynucleotides are useful model compounds for structural and biochemical studies on nucleic acids. Since a wide range of current molecular biological techniques rely on autoradiographic detection of gel elecrophoresis patterns, there is considerable demand for high-specific-activity 32Plabeled oligo- and polyribonucleotides. Effective methods exist for preparing end labeled materials (l-4), but some types of studies require uniformly labeled samples (5). These can be synthesized enzymatically from ribonucleoside 5’-[a-32P]diphosphates by use of polynucleotide phosphorylase (EC 2.7.7.8). Although ribonucleoside 5’-[cy-32P]diphosphates can be synthesized chemically, the procedures are laborious and time consuming. We present here a simple enzymatic precedure that takes less than an hour with nearly quantitative yield. Ribonucleoside 5’-[a-32P]triphosphates are readily available commercially. They can be converted into [a-32P]diphosphates irrevers-
ibly and completely within 10 min by myosin ATPase. This ATPase activity is a specialized function of the myosin molecule (6,7). Although named an ATPase, myosin utilizes all four ribonucleoside triphosphates. It removes only the terminal phosphate of the triphosphates, and does not dephosphorylate nucleoside diphosphates, monophosphates, or pyrophosphates. The enzyme is activated by Ca2’ and inhibited by Mg2+. Polynucleotide phosphorylase (PNPase)2 catalyzes the reversible polymerization of ribonucleoside diphosphates with release of inorganic phosphate in the presence of a divalent cation, Mg2+ or Mn2+ (8,9). Ribonucleoside 5’-[a-32P]diphosphates that are prepared by myosin ATPase can be used directly, without purification, by PNPase to form long-chain polymers in a few hours.
’ Financial support was provided by USPHS Grant GM 19843.
* Abbreviation used: PNPase, polynucleotide phorylase; PEI, polyethyleneimine.
MATERIALS
AND
METHODS
Materials. Rabbit muscle myosin was purchased from Sigma Chemical Company as a
291
phos-
0003-2697/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form resewed.
292
KANG
AND
solution in 50% glycerol containing 0.6 M KC1 (pH 6.8). Its concentration was 0.7 unit/ mg protein, or 7 units/ml solution, where 1 unit liberates 1 pmol inorganic phosphate from ATP/min at pH 9 and 25°C in the presence of Ca2+. Polynucleotide phosphorylase isolated from Micrococcus luteus, primer independent type, was purchased from Bethesda Research Laboratories, Inc., as a solution in 50% glycerol containing 10 mM Tris-HCl (pH 8.2), 1 mM MgC12, 1 mM 2-mercaptoethanol. The concentration was 800 units/ml, where 1 unit polymerizes 1 pmol ADP into poly(A) in 15 min at 37°C in a buffer solution consisting of 0.1 M Tris-HCl (pH 9) 5 tnM MgC12, 0.4 mM EDTA. Adenosine and uridine 5’-[a-32P]triphosphates were purchased from Amersham Corporation as triethylammonium salts in stabilized aqueous solution containing 5 mM 2mercaptoethanol. Radioactive concentration was 10 mCi/ml, and specific activity was 4 10 Ci/mmol at the reference date. PEI-cellulose TLC plates (Polygram CEL300PEI), 20 X 20 cm, manufactured by Macherey-Nagel Company, Diiren, Germany, were purchased from Brinkmann Instruments, Inc. These plastic sheets contain a O.l-mm layer of cellulose MN300 impregnated with polyethyleneimine (PEI). They were washed by ascending irrigation with distilled water. Impurities were transferred to a wick of thick absorbent paper stapled to the top of the sheet. After the sheets were dried at room temperature, several were wrapped together in aluminum foil and stored at -20°C.
CANTOR
Additional l-r1 aliquots were taken at various times from 2 to 90 min. Each aliquot was immediately spotted 2 cm from the bottom edge of a PEI-cellulose TLC plate using a micropipet. After drying the samples by a gentle stream of cool air, ascending chromatography was carried out with an acidic (pH 2.2) solvent mixture of 8:2 (v/v) 1 N acetic acid:4 M lithium chloride. In 2 h the solvent front migrated 16 cm. The developed TLC plates were dried in a current of cool air. Eastman-Kodak X-Omat AR X-ray films were exposed to the dried TLC plates without an intensifying screen for a few minutes at room temperature. A control PEI-cellulose TLC containing 5 nmol nonradioactive UTP, UDP, UMP, ATP, ADP, and AMP was used to identify particular species. The standards were detected using shortwave uv light. Preparative-scale 1O-p1 reactions were carried out for 15 min with 2.4 nmol lyophilized radioactive triphosphates (with no unlabeled carrier) in a 1.5-ml siliconized Eppendorf tube. For time-course PNPase polymerizations described below, lo-p1 reactions were carried out for 15 min with 2.4 pmol radioactive triphosphates and 2.2 nmol unlabeled ones. A 1.5~~1 aliquot of radioactive myosin reaction mixture was mixed with 8.5 ~1 blank myosin reaction buffer, consisting of 0.1 M glycine-NaOH (pH 9) and 10 mM CaC12. No effort to stop the reaction was necessary, and the entire reaction mixture was added directly to a 100~~1 PNPase reaction, as described below. Polymerization of ribonucleoside diphosphates by PNPase. Identical 100~~1 reactions
were used for time-course and preparative experiments. Each contained 0.1 M Tris-HCl 0.5 nmol [(u-~‘P]UTP or [c+~~P]ATP was (pH 8.6), 2 mM Mg(OAc)2, 5 mM nonralyophilized in a 1.5-ml siliconized Eppendorf dioactive UDP or ADP, and 2 units PNPase tube, and dissolved in 12 ~1 deionized-disin a 1.5-ml siliconized Eppendorf tube. To tilled water. This was mixed with 2 ~1 5 mM these were added entire lo-p1 ATPase reacunlabeled UTP or ATP, 2 ~1 0.1 M CaC12, 2 tions resulting in a carryover of 10 mM ~1 1 M glycine-NaOH (pH 9), and 2 ~1 glycine-NaOH (pH 9) and 1 mM CaC12. myosin ATPase (0.014 unit). Immediately a For time-course studies, PNPase reactions l-p1 aliquot was taken as a zero time point. were incubated at 37°C for l/2, 1, 2, 3, or 4 h, and immediately chilled on ice. The pH The reaction was started by 25°C incubation. Conversion of triphosphates to diphosphates by myosin ATPase. For time-course studies
ENZYMATIC
SYNTHESIS
OF RADIOACTIVE
was adjusted to 7.5 by adding 100 ~1 1 mM Tris-HCl (pH 7.5) and 5 ~1 1 N HCl. An aliquot of each RNA was extracted three times with 200 ~1 phenol:chloroform:isoamyl alcohol (25:24: 1, v/v/v). After residual organits were removed by ether extraction, NaOAc was added to 0.3 M. RNA was precipitated by 1 ml ethanol at -20°C overnight. Each radioactive sample was counted in lo-ml Liquiscint solution, a xylene- and toluenebased liquid scintillation cocktail, for 5 min on a Beckman LS3 133P counter. Preparative scale 100~~1 PNPase reactions were carried out for 1.5 h with the preparative-scale lo~1 myosin ATPase reaction described above. RESULTS
AND
293
POLYRIBONUCLEOTIDES
pletely hydrolyzed the terminal phosphate from both UTP and ATP in 10 min, as shown in Figs. 1 and 2. Nearly all the starting triphosphates were irreversibly converted to diphosphates. The PEI-cellulose TLC development system employed here separated all the nucleoside phosphates very well, and the apparent measured RJ values were close to the previously reported values ( 10). When the reaction was allowed to proceed for too long, some nucleoside monophosphates were seen, particularly after 30 min, probably due to some contaminating enzyme activities. However, the amounts of both monophosphates were not significant even after 1.5 h. CTP, GTP, and ITP are expected to follow roughly the identical time course, based on the known nonspecific characteristics of myosin ATPase (6,7). Under certain
DISCUSSION
Using the conditions described under Materials and Methods, myosin ATPase com-
UDP
UTP
Origin 0
2
5
10
20
30
45
60
90
min
FIG. 1. Time course for conversion of [w~~P]UTP to [a-32P]UDP by myosin ATPase. The TLC plate was developed with 0.8 N acetic acid and 0.8 M LiCl. The autoradiograph shows that the optimum reaction time is 10 to 20 min.
RANG
*r
AND CANTOR
ia ! sn _ -*t \\a
‘2
6_r
? ;”
:,” $2.
_I ($2,
3 \j
\’e; p,I
>?\b f‘,,,s
“3’ i ‘f
FIG. 2. Time course for conversion of [(u-~~P]ATP to [ a-32P]ADP by myosin ATPase. The TLC was developed as in Fig. 1. The autoradiograph shows that the optimum reaction time is 10 to 20 min.
conditions some triphosphates are even better substrates for the enzyme than ATP. The entire myosin ATPase reaction mixture was added intact as a substrate for PNPase. As shown in Fig. 3, polymerization reached maximum yield in 1 h using conditions described under Materials and Methods for both poly(A) and poly(U) syntheses. After 4 h, polymerization yields decreased significantly. At the beginning of the reaction the concentration of nucleoside diphosphate was much larger than the concentration of inorganic phosphate, which drives the reaction toward polymerization. As the concentration of inorganic phosphate increases, the backward phosphorolysis rate increased. Finally, a dynamic equilibrium between phosphorolysis and polymerization was reached, resulting in medium-size polyribonucleotides, assuming a freely dissociating enzyme-polynucleotide system (11).
50K
E 40K e
01 0
1
2 TIME,
3
4
hr
FIG. 3. Time course for PNPase synthesis of poly(U) (0) and poly(A) (0). The radioactivity in ethanol-precig itable polymer reached a maximum in 1 h.
ENZYMATIC
SYNTHESIS
OF RADIOACTIVE
The length of the product polynucleotides is assumed to reach a maximum as the yield reaches a maximum in 1 h. At this point about 60% of the initial nucleoside diphosphate is incorporated into ethanol-precipitable polymer. The radiolabeled polynucleotides are rather homogeneous in size. A denaturing polyacrylamide-urea gel showed that the length of the synthesized polymer was longer than 500 nucleotides. There should be no difficulty in synthesizing poly(C), poly(G), and various random copolymers from the appropriate diphosphates (8,12). The specific activity of the product polynucleotide ranged from 0.3 to 2 Ci/mmol residue depending on the molar ratio of radioactive and nonradioactive tri- and diphosphates used. No attempt to obtain higher specific activities was made, because 2 Ci/ mmol residue is equivalent to higher than 1000 Ci/mmol polymer for chains longer than 500 residues. This is high enough for most experiments. One can presumably increase it further, until degradation from radiochemical damage becomes a problem. REFERENCES 1. Richardson, C. C. (1982) in The Enzymes (Boyer, P. D. ed.), 3rd ed., Vol. 15, pp. 299-314, Academic Press, New York.
POLYRIBONUCLEOTIDES
295
2. Chaconas, G., and van de Sande, J. H. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 75-85, Academic Press, New York. 3. England, T. E., Bruce, A. G., and Uhlenbeck, 0. C. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 65-74, Academic Press, New York. 4. Sternbach, H., van de Haar, F., Schlimme, E., Gaertner, E., and Cramer, F. (197 1) Eur. J. Biochem.
22, 166-172.
5. Kang, C. (1983) Ph.D. Thesis, Columbia University Department of Chemistry, New York. 6. Kielley, W. W. (1961) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 5, pp. 159-168, Academic Press, New York. 7. Perry, S. V. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 582-588, Academic Press, New York. 8. Littauer, U. Z., and Soreq, H. (1982) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 15, pp. 5 18-553, Academic Press, New York. 9. Godefroy-Colbum, T., and Grunberg-Manago, M. (1972) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 7, pp. 533-574, Academic Press, New York. 10. Randerath, K., and Randerath, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, pp. 323-347, Academic Press, New York. 11. Cantor, C. R. (1968) Biopolymers 6, 369-384. 12. Seliger, H., and Kniible, T. (1978) Nucl. Acids Rex (Special publication) 4, s167-~170.
BIOCHEMISTRY
144,291-295
(1985)
Enzymatic Synthesis of Uniformly 32P-Labeled Polyribonucleotides High-Specific-Activity Ribonucleoside 5’-[a-32P]diphosphates’ R. CANTOR
CHANGWONKANGANDCHARLES Department
and
of Human Genetics and Development, College of Physicians Columbia University, New York, New York 10032
and Surgeons,
Received June 2 1, 1984 Uniformly “P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5’-[ru-32P]ttiphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition. 0 1985 Academic press, hc. KEY WORDS: nucleic acid chemistry; myosin ATPase; polynucleotide phosphorylase; poly(U); poly(A); nucleoside diphosphates.
Synthetic oligo- and polynucleotides are useful model compounds for structural and biochemical studies on nucleic acids. Since a wide range of current molecular biological techniques rely on autoradiographic detection of gel elecrophoresis patterns, there is considerable demand for high-specific-activity 32Plabeled oligo- and polyribonucleotides. Effective methods exist for preparing end labeled materials (l-4), but some types of studies require uniformly labeled samples (5). These can be synthesized enzymatically from ribonucleoside 5’-[a-32P]diphosphates by use of polynucleotide phosphorylase (EC 2.7.7.8). Although ribonucleoside 5’-[cy-32P]diphosphates can be synthesized chemically, the procedures are laborious and time consuming. We present here a simple enzymatic precedure that takes less than an hour with nearly quantitative yield. Ribonucleoside 5’-[a-32P]triphosphates are readily available commercially. They can be converted into [a-32P]diphosphates irrevers-
ibly and completely within 10 min by myosin ATPase. This ATPase activity is a specialized function of the myosin molecule (6,7). Although named an ATPase, myosin utilizes all four ribonucleoside triphosphates. It removes only the terminal phosphate of the triphosphates, and does not dephosphorylate nucleoside diphosphates, monophosphates, or pyrophosphates. The enzyme is activated by Ca2’ and inhibited by Mg2+. Polynucleotide phosphorylase (PNPase)2 catalyzes the reversible polymerization of ribonucleoside diphosphates with release of inorganic phosphate in the presence of a divalent cation, Mg2+ or Mn2+ (8,9). Ribonucleoside 5’-[a-32P]diphosphates that are prepared by myosin ATPase can be used directly, without purification, by PNPase to form long-chain polymers in a few hours.
’ Financial support was provided by USPHS Grant GM 19843.
* Abbreviation used: PNPase, polynucleotide phorylase; PEI, polyethyleneimine.
MATERIALS
AND
METHODS
Materials. Rabbit muscle myosin was purchased from Sigma Chemical Company as a
291
phos-
0003-2697/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form resewed.
292
KANG
AND
solution in 50% glycerol containing 0.6 M KC1 (pH 6.8). Its concentration was 0.7 unit/ mg protein, or 7 units/ml solution, where 1 unit liberates 1 pmol inorganic phosphate from ATP/min at pH 9 and 25°C in the presence of Ca2+. Polynucleotide phosphorylase isolated from Micrococcus luteus, primer independent type, was purchased from Bethesda Research Laboratories, Inc., as a solution in 50% glycerol containing 10 mM Tris-HCl (pH 8.2), 1 mM MgC12, 1 mM 2-mercaptoethanol. The concentration was 800 units/ml, where 1 unit polymerizes 1 pmol ADP into poly(A) in 15 min at 37°C in a buffer solution consisting of 0.1 M Tris-HCl (pH 9) 5 tnM MgC12, 0.4 mM EDTA. Adenosine and uridine 5’-[a-32P]triphosphates were purchased from Amersham Corporation as triethylammonium salts in stabilized aqueous solution containing 5 mM 2mercaptoethanol. Radioactive concentration was 10 mCi/ml, and specific activity was 4 10 Ci/mmol at the reference date. PEI-cellulose TLC plates (Polygram CEL300PEI), 20 X 20 cm, manufactured by Macherey-Nagel Company, Diiren, Germany, were purchased from Brinkmann Instruments, Inc. These plastic sheets contain a O.l-mm layer of cellulose MN300 impregnated with polyethyleneimine (PEI). They were washed by ascending irrigation with distilled water. Impurities were transferred to a wick of thick absorbent paper stapled to the top of the sheet. After the sheets were dried at room temperature, several were wrapped together in aluminum foil and stored at -20°C.
CANTOR
Additional l-r1 aliquots were taken at various times from 2 to 90 min. Each aliquot was immediately spotted 2 cm from the bottom edge of a PEI-cellulose TLC plate using a micropipet. After drying the samples by a gentle stream of cool air, ascending chromatography was carried out with an acidic (pH 2.2) solvent mixture of 8:2 (v/v) 1 N acetic acid:4 M lithium chloride. In 2 h the solvent front migrated 16 cm. The developed TLC plates were dried in a current of cool air. Eastman-Kodak X-Omat AR X-ray films were exposed to the dried TLC plates without an intensifying screen for a few minutes at room temperature. A control PEI-cellulose TLC containing 5 nmol nonradioactive UTP, UDP, UMP, ATP, ADP, and AMP was used to identify particular species. The standards were detected using shortwave uv light. Preparative-scale 1O-p1 reactions were carried out for 15 min with 2.4 nmol lyophilized radioactive triphosphates (with no unlabeled carrier) in a 1.5-ml siliconized Eppendorf tube. For time-course PNPase polymerizations described below, lo-p1 reactions were carried out for 15 min with 2.4 pmol radioactive triphosphates and 2.2 nmol unlabeled ones. A 1.5~~1 aliquot of radioactive myosin reaction mixture was mixed with 8.5 ~1 blank myosin reaction buffer, consisting of 0.1 M glycine-NaOH (pH 9) and 10 mM CaC12. No effort to stop the reaction was necessary, and the entire reaction mixture was added directly to a 100~~1 PNPase reaction, as described below. Polymerization of ribonucleoside diphosphates by PNPase. Identical 100~~1 reactions
were used for time-course and preparative experiments. Each contained 0.1 M Tris-HCl 0.5 nmol [(u-~‘P]UTP or [c+~~P]ATP was (pH 8.6), 2 mM Mg(OAc)2, 5 mM nonralyophilized in a 1.5-ml siliconized Eppendorf dioactive UDP or ADP, and 2 units PNPase tube, and dissolved in 12 ~1 deionized-disin a 1.5-ml siliconized Eppendorf tube. To tilled water. This was mixed with 2 ~1 5 mM these were added entire lo-p1 ATPase reacunlabeled UTP or ATP, 2 ~1 0.1 M CaC12, 2 tions resulting in a carryover of 10 mM ~1 1 M glycine-NaOH (pH 9), and 2 ~1 glycine-NaOH (pH 9) and 1 mM CaC12. myosin ATPase (0.014 unit). Immediately a For time-course studies, PNPase reactions l-p1 aliquot was taken as a zero time point. were incubated at 37°C for l/2, 1, 2, 3, or 4 h, and immediately chilled on ice. The pH The reaction was started by 25°C incubation. Conversion of triphosphates to diphosphates by myosin ATPase. For time-course studies
ENZYMATIC
SYNTHESIS
OF RADIOACTIVE
was adjusted to 7.5 by adding 100 ~1 1 mM Tris-HCl (pH 7.5) and 5 ~1 1 N HCl. An aliquot of each RNA was extracted three times with 200 ~1 phenol:chloroform:isoamyl alcohol (25:24: 1, v/v/v). After residual organits were removed by ether extraction, NaOAc was added to 0.3 M. RNA was precipitated by 1 ml ethanol at -20°C overnight. Each radioactive sample was counted in lo-ml Liquiscint solution, a xylene- and toluenebased liquid scintillation cocktail, for 5 min on a Beckman LS3 133P counter. Preparative scale 100~~1 PNPase reactions were carried out for 1.5 h with the preparative-scale lo~1 myosin ATPase reaction described above. RESULTS
AND
293
POLYRIBONUCLEOTIDES
pletely hydrolyzed the terminal phosphate from both UTP and ATP in 10 min, as shown in Figs. 1 and 2. Nearly all the starting triphosphates were irreversibly converted to diphosphates. The PEI-cellulose TLC development system employed here separated all the nucleoside phosphates very well, and the apparent measured RJ values were close to the previously reported values ( 10). When the reaction was allowed to proceed for too long, some nucleoside monophosphates were seen, particularly after 30 min, probably due to some contaminating enzyme activities. However, the amounts of both monophosphates were not significant even after 1.5 h. CTP, GTP, and ITP are expected to follow roughly the identical time course, based on the known nonspecific characteristics of myosin ATPase (6,7). Under certain
DISCUSSION
Using the conditions described under Materials and Methods, myosin ATPase com-
UDP
UTP
Origin 0
2
5
10
20
30
45
60
90
min
FIG. 1. Time course for conversion of [w~~P]UTP to [a-32P]UDP by myosin ATPase. The TLC plate was developed with 0.8 N acetic acid and 0.8 M LiCl. The autoradiograph shows that the optimum reaction time is 10 to 20 min.
RANG
*r
AND CANTOR
ia ! sn _ -*t \\a
‘2
6_r
? ;”
:,” $2.
_I ($2,
3 \j
\’e; p,I
>?\b f‘,,,s
“3’ i ‘f
FIG. 2. Time course for conversion of [(u-~~P]ATP to [ a-32P]ADP by myosin ATPase. The TLC was developed as in Fig. 1. The autoradiograph shows that the optimum reaction time is 10 to 20 min.
conditions some triphosphates are even better substrates for the enzyme than ATP. The entire myosin ATPase reaction mixture was added intact as a substrate for PNPase. As shown in Fig. 3, polymerization reached maximum yield in 1 h using conditions described under Materials and Methods for both poly(A) and poly(U) syntheses. After 4 h, polymerization yields decreased significantly. At the beginning of the reaction the concentration of nucleoside diphosphate was much larger than the concentration of inorganic phosphate, which drives the reaction toward polymerization. As the concentration of inorganic phosphate increases, the backward phosphorolysis rate increased. Finally, a dynamic equilibrium between phosphorolysis and polymerization was reached, resulting in medium-size polyribonucleotides, assuming a freely dissociating enzyme-polynucleotide system (11).
50K
E 40K e
01 0
1
2 TIME,
3
4
hr
FIG. 3. Time course for PNPase synthesis of poly(U) (0) and poly(A) (0). The radioactivity in ethanol-precig itable polymer reached a maximum in 1 h.
ENZYMATIC
SYNTHESIS
OF RADIOACTIVE
The length of the product polynucleotides is assumed to reach a maximum as the yield reaches a maximum in 1 h. At this point about 60% of the initial nucleoside diphosphate is incorporated into ethanol-precipitable polymer. The radiolabeled polynucleotides are rather homogeneous in size. A denaturing polyacrylamide-urea gel showed that the length of the synthesized polymer was longer than 500 nucleotides. There should be no difficulty in synthesizing poly(C), poly(G), and various random copolymers from the appropriate diphosphates (8,12). The specific activity of the product polynucleotide ranged from 0.3 to 2 Ci/mmol residue depending on the molar ratio of radioactive and nonradioactive tri- and diphosphates used. No attempt to obtain higher specific activities was made, because 2 Ci/ mmol residue is equivalent to higher than 1000 Ci/mmol polymer for chains longer than 500 residues. This is high enough for most experiments. One can presumably increase it further, until degradation from radiochemical damage becomes a problem. REFERENCES 1. Richardson, C. C. (1982) in The Enzymes (Boyer, P. D. ed.), 3rd ed., Vol. 15, pp. 299-314, Academic Press, New York.
POLYRIBONUCLEOTIDES
295
2. Chaconas, G., and van de Sande, J. H. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 75-85, Academic Press, New York. 3. England, T. E., Bruce, A. G., and Uhlenbeck, 0. C. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 65-74, Academic Press, New York. 4. Sternbach, H., van de Haar, F., Schlimme, E., Gaertner, E., and Cramer, F. (197 1) Eur. J. Biochem.
22, 166-172.
5. Kang, C. (1983) Ph.D. Thesis, Columbia University Department of Chemistry, New York. 6. Kielley, W. W. (1961) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 5, pp. 159-168, Academic Press, New York. 7. Perry, S. V. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 582-588, Academic Press, New York. 8. Littauer, U. Z., and Soreq, H. (1982) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 15, pp. 5 18-553, Academic Press, New York. 9. Godefroy-Colbum, T., and Grunberg-Manago, M. (1972) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 7, pp. 533-574, Academic Press, New York. 10. Randerath, K., and Randerath, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, pp. 323-347, Academic Press, New York. 11. Cantor, C. R. (1968) Biopolymers 6, 369-384. 12. Seliger, H., and Kniible, T. (1978) Nucl. Acids Rex (Special publication) 4, s167-~170.