Volume3 no.7 July1976
Nucleic Acids Research .M
Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.
L .David Beckman and Gerald D. Frenkel Department of Micrcbiology and Immunology, Neil Hellman Medical Research Building, Albany Medical College of Union University, Albany, NY 12208, USA.
Received 26 April 1976 ABSTRACT
A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure. INTRODUCTION
Electrophoresis in polyacrylamide gels under non-denaturing conditions has been a powerful method for the separation and analysis of proteins (1). However, the detection of DNA dependent DNA or RNA polymerase activities following electrophoresis in polyacrylamide gels has thus far depended on 1) elution of enzyme activity from gels sliced into discs, or 2) staining of the reaction product in the intact gel which contains template or template-primer. Both methods have severe limitations. In the slicing method, recovery of activity is variable and relatively large volumes of eluate are obtained which must often be concentrated. The staining method depends on the ability of the stain to detect product in the presence of template or template-primer. DNA polymerase I of E. coli can be detected by the staining method utilizing poly [d(A-T)] as template-primer because a minute amount of poly [d(A-T)] is sufficient to induce an extensive synthesis of poly [d(A-T)] in the gel by this enzyme (2). However, this property is not shared by the other known DNA polymerases and, therefore, the staining method cannot be used for the detection of these enzymes. Utilization of the staino Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1727
Nucleic Acids Research ing method for the detection of RNA polymerase has been limited to an enzyme from Azobacter capable of template independent polyribonucleotide synthesis (3). Attempts to detect template dependent synthesis by RNA polymerase from E. coli in intact polyacrylamide gels have given equivocal results (4). Jovin et al. (5) described an indirect procedure for detection of E. coli DNA polymerase I in gels, which utilizes radioactive precursors. In this report, we describe a new method for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels which also depends on the incorporation of radioactive deoxyribonucleoside or ribonucleoside triphosphate into the reaction product. This method is direct, quantitative, has high sensitivity and wide applicability, and few of the limitations of previously reported methods. EXPERIMENTAL PROCEDURE Materials Enzymes - Pancreatic DNase was purchased from Worthington. E. coli RNA polymerase, purified by the glycerol gradient procedure of Burgess (6), was a generous gift of Dr. R. Trimble. DNA - Salmon Sperm DNA was purchased from Sigma. Calf thymus DNA was purchased from Worthington. Activated DNA was prepared by treatment of calf thymus DNA with pancreatic DNase to 25% hyperchromicity. The DNase was inactivated by incubation at 600 for 30 min and the DNA was precipi-
tated with ethanol. Cells - KB cells (a permanent line of human fibroblasts) were grown in Dulbecco's Modified Eagle's medium containing 10% calf serum. Other Materials - [3H]TTP, [3H]UTP, [c_-32P]TTP, and [a-32P]UTP, as well as Protosol and Liquifluor were purchased from New England Nuclear. Unlabeled deoxyribonucleoside and ribonucleoside triphosphates were purchased from P. L. Biochemicals. Rifampin (B grade) and N-ethylmaleimide were purchased from Calbiochem. All chemicals used for electrophoresis were purchased from Bio-Rad Laboratories. Calf serum and Dulbecco's Modified Eagle's medium were purchased from Grand Island Biological Company. Methods
Preparation of KB Cell Extract - Confluent monolayers were washed once with 10 mM sodium phosphate buffer (pH 7.6) containing 0.15 M NaCl and the cells were removed from the plate with a rubber policeman and sus1728
Nucleic Acids Research pended in the same buffer. The cells were then centrifuged and resuspended in 10 mM potassium phosphate buffer (pH 7.6) containing 10 mM NaCl and 1 mM dithiothreitol at a cell density of approximately 4x107 the cells were homogenized with a cells/ml. After one hour at 0 dounce homogenizer, made 0.18 M in potassium phosphate buffer (pH 7.6), 9% in sucrose and 0.7 mM in MgCl2, and frozen at -20°. After thawing, the cells were sonically irradiated and centrifuged for 10 min at The supernatant is referred to as the KB cell extract. Ten 10,000g. 100 mm plates (approximately 3x107 cells) yielded 2 ml of extract containing approximately 10 mg/ml of protein and 0.25 units/ml of DNA polymerase activity (one unit of DNA polymerase activity is defined as the amount catalyzing the incorporation of 1 nmole of TTP in 30 min at ,
370). Electrophoresis - Electrophoresis under non-denaturing conditions was performed in a discontinuous system by a modification of the procedure of Ornstein and Davis (7) essentially as previously described (8). The stacking gel had the dimensions 0.6 x 1 cm and was photopolymerized from a solution containing 2.5% acrylamide and 0.6% bis-acrylamide in Tris-HCl buffer (pH 6.9). The separating gel had the dimensions 0.6 x 10 cm and was polymerized from a solution containing either 7.5% acrylamide and 0.2% bis-acrylamide or 5% acrylamide and 0.1% bis-acrylamide, as well as DNA, in Tris-HCl buffer (pH 8.9). For certain experiments DNA was omitted. Two volumes of sample were mixed with one volume of a solution of 40% sucrose containing 0.5% bromophenol blue as tracking dye and layered atop the stacking gel. Electrophoresis was begun at a current of 3 mA/gel. After 40 min, the current was increased to 5 mA/gel and electrophoresis was continued until the tracking dye reached the bottom of the separating gel. Electrophoresis was performed at 40 and precautions included immersing almost the entire gel tube in electrophoresis buffer (i.e., the upper half in cathode buffer and the lower half in anode buffer) in order to more readily dissipate heat. Detection of DNA or RNA Polymerase Activity - Following electrophoresis, the gel was removed from the gel tube, placed in a test tube, and sufficient reaction mixture was added to cover the gel (approximately 5 ml). For detection of DNA polymerase, the reaction mixture contained 50 mM Tris-HC1 buffer (pH 8.1), 10 mM MgCl2, 1.7 mM dithiothreitol, 50 mM KCI, 50 1M each of dATP, dCTP, dGTP and either 2.5 'PM (3H]TTP
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'4
2)
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40
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loo
SLICE NUMBER Figure 1. Detection of DNA polymerase activity in a KB cell extract following electrophoresis in a polyacrylamide gel: Analysis by slicing. 0.2 ml of a KB cell extract prepared as described under "Methods" was electrophoresed in a 7.5% polyacrylamide gel containing 70 pg/ml activated calf thymus DNA as described under "Methods". Following electrophoresis, the gel was incubated with DNA polymerase reaction mixture containing [3H]TTP for 90 min at 370 and analyzed by slicing as described under "Methods".
[oe-32P]TTP (3.5 Ci/mmole). For detection of (6.5 Ci/mmole) or 2.5 RNA polymerase, the reaction mixture contained 50 mM Tris-HCl buffer (pH 8.1), 10 mM MgCl2, 20 iM dithiofhzeitol, 50 mM KC1, 600 11M each 50 vIM [3H]UTP (0.25 Ci mmole) ATP, CTP, GTP, and either 50 }iM [a-32P]UTP (0.2 Ci/mmole). One unit of RNA polymerase activity is defined as the amount catalyzing the incorporation of 1 nmole of UTP or
in 30 min at 37
0
After incubation at 370, the reaction mixture was removed and the gel rinsed twice with cold 10% trichloroacetic acid. The gel was then placed in one of the tubes of a Bio-Rad gel destainer containing an activated charcoal cartridge and background radioactivity was removed
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2iii 0A
_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Sl.!
Figure 2. Detection of DNA polymerase activity in a KB cell extract following electrophoresis in a polyacrylamide gel: Analysis by autoradiography. The procedure was exactly as described in the legend to Figure 1, except that the reaction mixture contained [o1_32P]TTP in place of [3H]TTP. Autoradiography was performed as described under "Methods" and exposure time was 4 hours. The autoradiogram was scanned with a Beckman Model R-112 microdensitometer and the tracing obtained is aligned in register below the autoradiogram.
by diffusion into 6% trichloroacetic acid containing 0.1 M sodium pyrophosphate for 18 to 48 hours. The gel was then analyzed for radioactivity by either of the following two methods: 1) slicing; the gel was sliced into 1 mm discs, each disc was placed into a scintillation vial, covered with 0.3 ml of 90% Protosol and incubated overnight at room temperature. Four ml of liquid scintillator (40 ml Liquifluor per liter of toluene) were then added and the samples counted in a liquid scintillation counter, or 2) autoradiography; the gel was sliced longitudinally in half, the slices were dried and autoradiography was performed according to the method of Fairbanks et al. (9) using Kodak NS54T x-ray film. RESULTS
Analysis of DNA Polymerase in Intact Polyacrylamide Gels Detection of DNA Polymerase - A KB cell extract was subjected to electrophoresis in 7.5% polyacrylamide gels containing activated calf thymus DNA. Following electrophoresis, the gels were analyzed as described under "Methods". Figure 1 shows that a peak of radioactivity is observed after incubation of the gel with reaction mixture containing [3H]TTP and analysis by slicing. Figure 2 shows that a band is observed 1731
Nucleic Acids Research Table I. Deoxyribonuclease sensitivity of the band of radioactivity observed after incubation of an intact polyacrylamide gel with DNA - polymerase reaction mixture. Treatment
Acid precipitable 32p cpm
No incubation Incubation -DNase Incubation +DNase
12,745 10,544 961
% 100 83 8
In a gel similar to that shown in Figure 2, the location of the band was determined by autoradiography. This portion of the dried gel was cut out (approximately 1 cm in length) and placed into 1 ml of 1 N NaOH. After incubation overnight at room temperature, the liquid was removed and neutralized with 1 N HC1. All of the eluted radioactivity (>100%) was found to be insoluble in trichloroacetic acid. Aliquots (0.2 ml containing a total of 9,600 cpm) were added to a reaction mixture (0.3 ml) containing 50 mM Tris-HCl buffer (pH 7.6), 4 mM MgCl2, and 5 ig pancreatic DNase where indicated. Incubation was for 20 min at 370. Following incubation, 50 ig of salmon sperm DNA and trichloroacetic acid (to a final concentration of 5%) were added. In the control tube (no incubation), carrier DNA and trichloroacetic acid were added without the addition of DNase or prior incubation at 370. The mixtures were filtered through Whatman GF/C glass fiber filters. The filters were washed with 6% trichloroacetic acid containing 0.1 M sodium pyrophosphate, dried, and the radioactivity determined in a liquid scintillation counter.
after incubation of the gel with reaction mixture containing [a--32P]TTP and analysis by autoradiography. Analysis of the Reaction Product - In order to demonstrate that the trichloroacetic acid insoluble radioactivity remaining associated with the gels in Figures 1 and 2 is indeed due to DNA polymerase activity and not, for example, an artifact due to preferential trapping of radioactivity at a particular location on the gel, the product of the reaction was analyzed. The band region of a dried gel similar to that shown in Figure 2 was cut out of the gel and the radioactivity was eluted by incubation overnight with 1 N NaOH at room temperature. After neutralization of the NaOH with HC1, the eluted radioactivity was analyzed for its susceptibility to hydrolysis by DNase (Table I). In the absence of treatment with DNase, all of the radioactivity is insoluble in trichloroacetic acid. However, treatment with DNase renders more than 90% of this radioactivity soluble in trichloroacetic acid. This demonstrates that the radioactivity in the band of Figure 2, and by impli-
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Nucleic Acids Research
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Figure 3. Inhibition of KB cell DNA polymerase activity by N-ethylmaleimide. Electrophoresis of the KB cell extract and detection of DNA polymerase activity in intact gels, was performed as described in the legend to Figure 2, except that following electrophoresis the gels were pre-incubated for 5 min at 370 with 10 mM potassium phosphate buffer (pH 7.6) (A), or with the same buffer containing 20 mM N-ethylmaleimide (B). Reaction mixtures were as described under "Methods", except that the reaction mixture for gel B contained 20 mM N-ethylmaleimide and incubation was for 60 min.
cation in the peak of Figure 1, is in DNA and, therefore, must be the product of a reaction catalyzed by a DNA polymerase present in the KB cell extract. Template Dependence of Activity - If activated DNA is omitted from the gel in experiments such as those shown in Figures 1 and 2, DNA polymerase activity is still observed. However, if prior to electrophoresis the KB cell extract is incubated with 0.2 pg/ml pancreatic DNase for 10 1733
Nucleic Acids Research
-RIF
B +RIF|l
Figure 4. Detection of RNA polymerase activity and its inhibition by rifampin after electrophoresis in polyacrylamide gels. 0.7 units of purified E. coli RNA polymerase was electrophoresed in 5% polyacrylamide gels containing 100 1g/ml calf thymus DNA as described under "Methods". Following electrophoresis, the gels were pre-incubated at 370 with 10 mM Tris-HCl buffer (pH 8.1) (A), or the same buffer containing 20 Jg/ml rifampin (B). After 10 min, the gels were placed in RNA polymerase reaction mixture containing [a-32P]UTP (A) or in RNA polymerase reaction mixture containing 15 ig/ml rifampin and [a-32p]UTP (B). Following incubation for 1 hour at 370, autoradiography was performed as described under "Methods". Exposure time was 17 hours. Microdensitometer tracings were obtained as described in the legend to Figure 2. min at 370, then activity is observed only in gels which contain DNA. This result suggests that the KB cell extract contains fragmented DNA which is able to penetrate the gel during electrophoresis, possibly as a result of binding to the enzyme. Characterization of the DNA Polymerase Activity - The work of Sedwick et al. (10) has demonstrated the presence of two major DNA polymerases in KB cells, designated aand f. Only the a-polymerase is sensitive to
inhibition by sulfhydral reagents such as N-ethylmaleimide (10). Figure 3 shows that the band of DNA polymerase activity (indicated by the arrow) which is detected after electrophoresis of KB cell extracts is inhibited by N-ethylmaleimide and we conclude, therefore, that this activity represents the a-polymerase. The 1-polymerase is not detected in the gel, confirming the findings of others that the a-polymerase does not enter polyacrylamide gels under non-denaturing conditions below pH 9 (11)*. The activity at the origin is also inhibited by N-ethyl-
*Knopf, K.-W. (Roche Institute of Molecular Biology), personal communication. 1734
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20 40 60 80 100 20 40 60 80 100 SLICE NUMBER Figure 5. Detection in polyacrylamide gels of increasing amounts of electrophoresed enzyme. Purified E. coli RNA polymerase was electrophoresed in 5% polyacrylamide gels containing 100 pg/ml calf thymus DNA as described under "Methods". Following electrophoresis, the gels were incubated for 1 hour at 370 with RNA polymerase reaction mixture containing [3H]UTP and analyzed by slicing as described under "Methods". In (A) 1.4 units of enzyme activity, and in (B) 2.8 units of enzyme activity, were electrophoresed.
maleimide and hence probably represents the a-polymerase in its aggregated form(s) (12). Analysis of RNA Polymerase in Intact Polyacrylamide Gels Detection of RNA Polymerase - Purified E. coli RNA polymerase was
electrophoresed in a 5% polyacrylamide gel containing calf thymus DNA. The gel was assayed for RNA polymerase activity using the reaction mixture described under "Methods" and analyzed by autoradiography. Figure 4A shows that two peaks of RNA polymerase activity are observed in the microdensitometer tracing of the autoradiogram. The presence of multiple peaks is probably the result of aggregation of the enzyme (13). If DNA is omitted from the gel, no RNA polymerase activity can be detected. Characterization of the RNA Polymerase Activity - RNA synthesis by the E. coli RNA polymerase is known to be inhibited by rifampin (14). Figure 4B shows that in the presence of rifampin no peak is observed in the microdensitometer tracing of the autoradiogram. We therefore conclude that the radioactive peaks in Figure 4A are the result of RNA synthesis catalyzed by different aggregated forms of the E. coli RNA
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Nucleic Acids Research polymerase in the intact polyacrylamide gel. The experiment in Figure 5 shows that the RNA polymerase activity detected in the gel is approximately proportional to the amount of enzyme electrophoresed. Thus approximately twice as much activity is detected when 2.8 units of enzyme are electrophoresed (Fig. 5B) as when 1.4 units of enzyme are electrophoresed (Fig. 5A). DISCUSSION The results we have presented demonstrate that DNA and RNA polymerase can be detected in intact polyacrylamide gels, that these enzymes can be characterized by the use of inhibitors such as N-ethylmaleimide and rifampin, and that the recovery of activity is approximately proportional to the amount of enzyme electrophoresed. An important application of the procedure we have described is in the analysis of specific enzymes in crude systems containing multiple proteins with similar or identical enzymatic activities. The procedure should permit detection of minor enzymatic components in the presence of a large excess of enzymes with similar activity, and also permit a rapid screening for mutants defective in one of these enzymes. For this purpose, the procedure is currently being adapted for use in slab gels. The new procedure we have described can, in principle, be applied to any enzyme for which the radioactive product remains associated with the gel while the radioactive reactant does not. In the case of the polymerases this is a result of the polymeric nature of the product, as compared to the monomeric nature of the reactant. In addition, many low molecular weight compounds such as nucleosides, can be linked to high molecular weight molecules such as dextran and Ficoll (15), and it may be possible to incorporate such linked molecules into polyacrylamide gels. It may then be feasible to assay enzymes such as nucleoside kinases by this method, since radioactivity transferred to this linked substrate via an enzymatic reaction would remain associated with the gels, whereas unreacted radioactivity would not. These possibilities are currently under investigation.
ACKNOWLEDGEMENTS We thank Cynthia Searles for expert technical assistance, L. A. Caliguiri and C. C. Richardson for helpful discussions, and Kathy
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Nucleic Acids Research Benedetto for expert typing of the manuscript. This investigation was supported in part by U.S. Public Health Service Grant No. AI-11913 and by American Cancer Society Grant No. NP-198. REFERENCES 1. 2. 3.
4. 5.
6. 7. 8. 9.
10. 11. 12.
13. 14. 15.
Maurer, H.R. (1971). Disc Electrophoresis, deGruyter, Berlin. Neuhoff, V. and Lezius, A. (1968). Z. Naturforschung 23b, 812-819. Krakow, J.S., Daley, K. and Fronk, E. (1968). Biochem. Biophys. Res. Comm. 32, 98-104. Neuhoff, V., Schill, W. and Sternbach, H. (1968). Hoppe-Seyler's Z. Physiol. Chem. 349, 1126-1136. Jovin, T.M., Englund, P.T. and Bertsch, L.L. (1969). J. Biol. Chem. 244, 2996-3008. Burgess, R. (1969). J. Biol. Chem. 244, 6160-6167. Ornstein, L. (1964). Annals N.Y. Acad. Sci. 121, 321-349; Davis, B.J. (1964). ibid 121, 404-427. Beckman, L.D., Hoffman, M.S. and McCorquodale, D.J. (1971). J. Mol. Biol. 62, 551-564. Fairbanks, G., Levinthal, C. and Reeder, R.H. (1965). Biochem. Biophys. Res. Comm. 20, 393-399. Sedwick, W.D., Wang, T.S. and Korn, D. (1972). J. Biol. Chem. 247, 5026-5033. Greene, R. and Korn, D. (1970). J. Biol. Chem. 245, 254-261. Sedwick, W.D., Wang, T.S. and Korn, D. (1975). J. Biol. Chem. 250, 7045-7056. Richardson, J.P. (1969). Prog. Nucl. Acid Res. 9, 75-116. Hartmann, G., Honikel, K.O., Knisel, F. and Nuesch, J. (1967). Biochim. Biophys. Acta 145, 843-844. Scheffler, I.E. and Richardson, C.C. (1972). J. Biol. Chem.
247, 5736-5745.
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Nucleic Acids Research .M
Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.
L .David Beckman and Gerald D. Frenkel Department of Micrcbiology and Immunology, Neil Hellman Medical Research Building, Albany Medical College of Union University, Albany, NY 12208, USA.
Received 26 April 1976 ABSTRACT
A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure. INTRODUCTION
Electrophoresis in polyacrylamide gels under non-denaturing conditions has been a powerful method for the separation and analysis of proteins (1). However, the detection of DNA dependent DNA or RNA polymerase activities following electrophoresis in polyacrylamide gels has thus far depended on 1) elution of enzyme activity from gels sliced into discs, or 2) staining of the reaction product in the intact gel which contains template or template-primer. Both methods have severe limitations. In the slicing method, recovery of activity is variable and relatively large volumes of eluate are obtained which must often be concentrated. The staining method depends on the ability of the stain to detect product in the presence of template or template-primer. DNA polymerase I of E. coli can be detected by the staining method utilizing poly [d(A-T)] as template-primer because a minute amount of poly [d(A-T)] is sufficient to induce an extensive synthesis of poly [d(A-T)] in the gel by this enzyme (2). However, this property is not shared by the other known DNA polymerases and, therefore, the staining method cannot be used for the detection of these enzymes. Utilization of the staino Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1727
Nucleic Acids Research ing method for the detection of RNA polymerase has been limited to an enzyme from Azobacter capable of template independent polyribonucleotide synthesis (3). Attempts to detect template dependent synthesis by RNA polymerase from E. coli in intact polyacrylamide gels have given equivocal results (4). Jovin et al. (5) described an indirect procedure for detection of E. coli DNA polymerase I in gels, which utilizes radioactive precursors. In this report, we describe a new method for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels which also depends on the incorporation of radioactive deoxyribonucleoside or ribonucleoside triphosphate into the reaction product. This method is direct, quantitative, has high sensitivity and wide applicability, and few of the limitations of previously reported methods. EXPERIMENTAL PROCEDURE Materials Enzymes - Pancreatic DNase was purchased from Worthington. E. coli RNA polymerase, purified by the glycerol gradient procedure of Burgess (6), was a generous gift of Dr. R. Trimble. DNA - Salmon Sperm DNA was purchased from Sigma. Calf thymus DNA was purchased from Worthington. Activated DNA was prepared by treatment of calf thymus DNA with pancreatic DNase to 25% hyperchromicity. The DNase was inactivated by incubation at 600 for 30 min and the DNA was precipi-
tated with ethanol. Cells - KB cells (a permanent line of human fibroblasts) were grown in Dulbecco's Modified Eagle's medium containing 10% calf serum. Other Materials - [3H]TTP, [3H]UTP, [c_-32P]TTP, and [a-32P]UTP, as well as Protosol and Liquifluor were purchased from New England Nuclear. Unlabeled deoxyribonucleoside and ribonucleoside triphosphates were purchased from P. L. Biochemicals. Rifampin (B grade) and N-ethylmaleimide were purchased from Calbiochem. All chemicals used for electrophoresis were purchased from Bio-Rad Laboratories. Calf serum and Dulbecco's Modified Eagle's medium were purchased from Grand Island Biological Company. Methods
Preparation of KB Cell Extract - Confluent monolayers were washed once with 10 mM sodium phosphate buffer (pH 7.6) containing 0.15 M NaCl and the cells were removed from the plate with a rubber policeman and sus1728
Nucleic Acids Research pended in the same buffer. The cells were then centrifuged and resuspended in 10 mM potassium phosphate buffer (pH 7.6) containing 10 mM NaCl and 1 mM dithiothreitol at a cell density of approximately 4x107 the cells were homogenized with a cells/ml. After one hour at 0 dounce homogenizer, made 0.18 M in potassium phosphate buffer (pH 7.6), 9% in sucrose and 0.7 mM in MgCl2, and frozen at -20°. After thawing, the cells were sonically irradiated and centrifuged for 10 min at The supernatant is referred to as the KB cell extract. Ten 10,000g. 100 mm plates (approximately 3x107 cells) yielded 2 ml of extract containing approximately 10 mg/ml of protein and 0.25 units/ml of DNA polymerase activity (one unit of DNA polymerase activity is defined as the amount catalyzing the incorporation of 1 nmole of TTP in 30 min at ,
370). Electrophoresis - Electrophoresis under non-denaturing conditions was performed in a discontinuous system by a modification of the procedure of Ornstein and Davis (7) essentially as previously described (8). The stacking gel had the dimensions 0.6 x 1 cm and was photopolymerized from a solution containing 2.5% acrylamide and 0.6% bis-acrylamide in Tris-HCl buffer (pH 6.9). The separating gel had the dimensions 0.6 x 10 cm and was polymerized from a solution containing either 7.5% acrylamide and 0.2% bis-acrylamide or 5% acrylamide and 0.1% bis-acrylamide, as well as DNA, in Tris-HCl buffer (pH 8.9). For certain experiments DNA was omitted. Two volumes of sample were mixed with one volume of a solution of 40% sucrose containing 0.5% bromophenol blue as tracking dye and layered atop the stacking gel. Electrophoresis was begun at a current of 3 mA/gel. After 40 min, the current was increased to 5 mA/gel and electrophoresis was continued until the tracking dye reached the bottom of the separating gel. Electrophoresis was performed at 40 and precautions included immersing almost the entire gel tube in electrophoresis buffer (i.e., the upper half in cathode buffer and the lower half in anode buffer) in order to more readily dissipate heat. Detection of DNA or RNA Polymerase Activity - Following electrophoresis, the gel was removed from the gel tube, placed in a test tube, and sufficient reaction mixture was added to cover the gel (approximately 5 ml). For detection of DNA polymerase, the reaction mixture contained 50 mM Tris-HC1 buffer (pH 8.1), 10 mM MgCl2, 1.7 mM dithiothreitol, 50 mM KCI, 50 1M each of dATP, dCTP, dGTP and either 2.5 'PM (3H]TTP
1729
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'4
2)
20
40
60
80
loo
SLICE NUMBER Figure 1. Detection of DNA polymerase activity in a KB cell extract following electrophoresis in a polyacrylamide gel: Analysis by slicing. 0.2 ml of a KB cell extract prepared as described under "Methods" was electrophoresed in a 7.5% polyacrylamide gel containing 70 pg/ml activated calf thymus DNA as described under "Methods". Following electrophoresis, the gel was incubated with DNA polymerase reaction mixture containing [3H]TTP for 90 min at 370 and analyzed by slicing as described under "Methods".
[oe-32P]TTP (3.5 Ci/mmole). For detection of (6.5 Ci/mmole) or 2.5 RNA polymerase, the reaction mixture contained 50 mM Tris-HCl buffer (pH 8.1), 10 mM MgCl2, 20 iM dithiofhzeitol, 50 mM KC1, 600 11M each 50 vIM [3H]UTP (0.25 Ci mmole) ATP, CTP, GTP, and either 50 }iM [a-32P]UTP (0.2 Ci/mmole). One unit of RNA polymerase activity is defined as the amount catalyzing the incorporation of 1 nmole of UTP or
in 30 min at 37
0
After incubation at 370, the reaction mixture was removed and the gel rinsed twice with cold 10% trichloroacetic acid. The gel was then placed in one of the tubes of a Bio-Rad gel destainer containing an activated charcoal cartridge and background radioactivity was removed
1730
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2iii 0A
_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Sl.!
Figure 2. Detection of DNA polymerase activity in a KB cell extract following electrophoresis in a polyacrylamide gel: Analysis by autoradiography. The procedure was exactly as described in the legend to Figure 1, except that the reaction mixture contained [o1_32P]TTP in place of [3H]TTP. Autoradiography was performed as described under "Methods" and exposure time was 4 hours. The autoradiogram was scanned with a Beckman Model R-112 microdensitometer and the tracing obtained is aligned in register below the autoradiogram.
by diffusion into 6% trichloroacetic acid containing 0.1 M sodium pyrophosphate for 18 to 48 hours. The gel was then analyzed for radioactivity by either of the following two methods: 1) slicing; the gel was sliced into 1 mm discs, each disc was placed into a scintillation vial, covered with 0.3 ml of 90% Protosol and incubated overnight at room temperature. Four ml of liquid scintillator (40 ml Liquifluor per liter of toluene) were then added and the samples counted in a liquid scintillation counter, or 2) autoradiography; the gel was sliced longitudinally in half, the slices were dried and autoradiography was performed according to the method of Fairbanks et al. (9) using Kodak NS54T x-ray film. RESULTS
Analysis of DNA Polymerase in Intact Polyacrylamide Gels Detection of DNA Polymerase - A KB cell extract was subjected to electrophoresis in 7.5% polyacrylamide gels containing activated calf thymus DNA. Following electrophoresis, the gels were analyzed as described under "Methods". Figure 1 shows that a peak of radioactivity is observed after incubation of the gel with reaction mixture containing [3H]TTP and analysis by slicing. Figure 2 shows that a band is observed 1731
Nucleic Acids Research Table I. Deoxyribonuclease sensitivity of the band of radioactivity observed after incubation of an intact polyacrylamide gel with DNA - polymerase reaction mixture. Treatment
Acid precipitable 32p cpm
No incubation Incubation -DNase Incubation +DNase
12,745 10,544 961
% 100 83 8
In a gel similar to that shown in Figure 2, the location of the band was determined by autoradiography. This portion of the dried gel was cut out (approximately 1 cm in length) and placed into 1 ml of 1 N NaOH. After incubation overnight at room temperature, the liquid was removed and neutralized with 1 N HC1. All of the eluted radioactivity (>100%) was found to be insoluble in trichloroacetic acid. Aliquots (0.2 ml containing a total of 9,600 cpm) were added to a reaction mixture (0.3 ml) containing 50 mM Tris-HCl buffer (pH 7.6), 4 mM MgCl2, and 5 ig pancreatic DNase where indicated. Incubation was for 20 min at 370. Following incubation, 50 ig of salmon sperm DNA and trichloroacetic acid (to a final concentration of 5%) were added. In the control tube (no incubation), carrier DNA and trichloroacetic acid were added without the addition of DNase or prior incubation at 370. The mixtures were filtered through Whatman GF/C glass fiber filters. The filters were washed with 6% trichloroacetic acid containing 0.1 M sodium pyrophosphate, dried, and the radioactivity determined in a liquid scintillation counter.
after incubation of the gel with reaction mixture containing [a--32P]TTP and analysis by autoradiography. Analysis of the Reaction Product - In order to demonstrate that the trichloroacetic acid insoluble radioactivity remaining associated with the gels in Figures 1 and 2 is indeed due to DNA polymerase activity and not, for example, an artifact due to preferential trapping of radioactivity at a particular location on the gel, the product of the reaction was analyzed. The band region of a dried gel similar to that shown in Figure 2 was cut out of the gel and the radioactivity was eluted by incubation overnight with 1 N NaOH at room temperature. After neutralization of the NaOH with HC1, the eluted radioactivity was analyzed for its susceptibility to hydrolysis by DNase (Table I). In the absence of treatment with DNase, all of the radioactivity is insoluble in trichloroacetic acid. However, treatment with DNase renders more than 90% of this radioactivity soluble in trichloroacetic acid. This demonstrates that the radioactivity in the band of Figure 2, and by impli-
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Figure 3. Inhibition of KB cell DNA polymerase activity by N-ethylmaleimide. Electrophoresis of the KB cell extract and detection of DNA polymerase activity in intact gels, was performed as described in the legend to Figure 2, except that following electrophoresis the gels were pre-incubated for 5 min at 370 with 10 mM potassium phosphate buffer (pH 7.6) (A), or with the same buffer containing 20 mM N-ethylmaleimide (B). Reaction mixtures were as described under "Methods", except that the reaction mixture for gel B contained 20 mM N-ethylmaleimide and incubation was for 60 min.
cation in the peak of Figure 1, is in DNA and, therefore, must be the product of a reaction catalyzed by a DNA polymerase present in the KB cell extract. Template Dependence of Activity - If activated DNA is omitted from the gel in experiments such as those shown in Figures 1 and 2, DNA polymerase activity is still observed. However, if prior to electrophoresis the KB cell extract is incubated with 0.2 pg/ml pancreatic DNase for 10 1733
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-RIF
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Figure 4. Detection of RNA polymerase activity and its inhibition by rifampin after electrophoresis in polyacrylamide gels. 0.7 units of purified E. coli RNA polymerase was electrophoresed in 5% polyacrylamide gels containing 100 1g/ml calf thymus DNA as described under "Methods". Following electrophoresis, the gels were pre-incubated at 370 with 10 mM Tris-HCl buffer (pH 8.1) (A), or the same buffer containing 20 Jg/ml rifampin (B). After 10 min, the gels were placed in RNA polymerase reaction mixture containing [a-32P]UTP (A) or in RNA polymerase reaction mixture containing 15 ig/ml rifampin and [a-32p]UTP (B). Following incubation for 1 hour at 370, autoradiography was performed as described under "Methods". Exposure time was 17 hours. Microdensitometer tracings were obtained as described in the legend to Figure 2. min at 370, then activity is observed only in gels which contain DNA. This result suggests that the KB cell extract contains fragmented DNA which is able to penetrate the gel during electrophoresis, possibly as a result of binding to the enzyme. Characterization of the DNA Polymerase Activity - The work of Sedwick et al. (10) has demonstrated the presence of two major DNA polymerases in KB cells, designated aand f. Only the a-polymerase is sensitive to
inhibition by sulfhydral reagents such as N-ethylmaleimide (10). Figure 3 shows that the band of DNA polymerase activity (indicated by the arrow) which is detected after electrophoresis of KB cell extracts is inhibited by N-ethylmaleimide and we conclude, therefore, that this activity represents the a-polymerase. The 1-polymerase is not detected in the gel, confirming the findings of others that the a-polymerase does not enter polyacrylamide gels under non-denaturing conditions below pH 9 (11)*. The activity at the origin is also inhibited by N-ethyl-
*Knopf, K.-W. (Roche Institute of Molecular Biology), personal communication. 1734
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20 40 60 80 100 20 40 60 80 100 SLICE NUMBER Figure 5. Detection in polyacrylamide gels of increasing amounts of electrophoresed enzyme. Purified E. coli RNA polymerase was electrophoresed in 5% polyacrylamide gels containing 100 pg/ml calf thymus DNA as described under "Methods". Following electrophoresis, the gels were incubated for 1 hour at 370 with RNA polymerase reaction mixture containing [3H]UTP and analyzed by slicing as described under "Methods". In (A) 1.4 units of enzyme activity, and in (B) 2.8 units of enzyme activity, were electrophoresed.
maleimide and hence probably represents the a-polymerase in its aggregated form(s) (12). Analysis of RNA Polymerase in Intact Polyacrylamide Gels Detection of RNA Polymerase - Purified E. coli RNA polymerase was
electrophoresed in a 5% polyacrylamide gel containing calf thymus DNA. The gel was assayed for RNA polymerase activity using the reaction mixture described under "Methods" and analyzed by autoradiography. Figure 4A shows that two peaks of RNA polymerase activity are observed in the microdensitometer tracing of the autoradiogram. The presence of multiple peaks is probably the result of aggregation of the enzyme (13). If DNA is omitted from the gel, no RNA polymerase activity can be detected. Characterization of the RNA Polymerase Activity - RNA synthesis by the E. coli RNA polymerase is known to be inhibited by rifampin (14). Figure 4B shows that in the presence of rifampin no peak is observed in the microdensitometer tracing of the autoradiogram. We therefore conclude that the radioactive peaks in Figure 4A are the result of RNA synthesis catalyzed by different aggregated forms of the E. coli RNA
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Nucleic Acids Research polymerase in the intact polyacrylamide gel. The experiment in Figure 5 shows that the RNA polymerase activity detected in the gel is approximately proportional to the amount of enzyme electrophoresed. Thus approximately twice as much activity is detected when 2.8 units of enzyme are electrophoresed (Fig. 5B) as when 1.4 units of enzyme are electrophoresed (Fig. 5A). DISCUSSION The results we have presented demonstrate that DNA and RNA polymerase can be detected in intact polyacrylamide gels, that these enzymes can be characterized by the use of inhibitors such as N-ethylmaleimide and rifampin, and that the recovery of activity is approximately proportional to the amount of enzyme electrophoresed. An important application of the procedure we have described is in the analysis of specific enzymes in crude systems containing multiple proteins with similar or identical enzymatic activities. The procedure should permit detection of minor enzymatic components in the presence of a large excess of enzymes with similar activity, and also permit a rapid screening for mutants defective in one of these enzymes. For this purpose, the procedure is currently being adapted for use in slab gels. The new procedure we have described can, in principle, be applied to any enzyme for which the radioactive product remains associated with the gel while the radioactive reactant does not. In the case of the polymerases this is a result of the polymeric nature of the product, as compared to the monomeric nature of the reactant. In addition, many low molecular weight compounds such as nucleosides, can be linked to high molecular weight molecules such as dextran and Ficoll (15), and it may be possible to incorporate such linked molecules into polyacrylamide gels. It may then be feasible to assay enzymes such as nucleoside kinases by this method, since radioactivity transferred to this linked substrate via an enzymatic reaction would remain associated with the gels, whereas unreacted radioactivity would not. These possibilities are currently under investigation.
ACKNOWLEDGEMENTS We thank Cynthia Searles for expert technical assistance, L. A. Caliguiri and C. C. Richardson for helpful discussions, and Kathy
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Nucleic Acids Research Benedetto for expert typing of the manuscript. This investigation was supported in part by U.S. Public Health Service Grant No. AI-11913 and by American Cancer Society Grant No. NP-198. REFERENCES 1. 2. 3.
4. 5.
6. 7. 8. 9.
10. 11. 12.
13. 14. 15.
Maurer, H.R. (1971). Disc Electrophoresis, deGruyter, Berlin. Neuhoff, V. and Lezius, A. (1968). Z. Naturforschung 23b, 812-819. Krakow, J.S., Daley, K. and Fronk, E. (1968). Biochem. Biophys. Res. Comm. 32, 98-104. Neuhoff, V., Schill, W. and Sternbach, H. (1968). Hoppe-Seyler's Z. Physiol. Chem. 349, 1126-1136. Jovin, T.M., Englund, P.T. and Bertsch, L.L. (1969). J. Biol. Chem. 244, 2996-3008. Burgess, R. (1969). J. Biol. Chem. 244, 6160-6167. Ornstein, L. (1964). Annals N.Y. Acad. Sci. 121, 321-349; Davis, B.J. (1964). ibid 121, 404-427. Beckman, L.D., Hoffman, M.S. and McCorquodale, D.J. (1971). J. Mol. Biol. 62, 551-564. Fairbanks, G., Levinthal, C. and Reeder, R.H. (1965). Biochem. Biophys. Res. Comm. 20, 393-399. Sedwick, W.D., Wang, T.S. and Korn, D. (1972). J. Biol. Chem. 247, 5026-5033. Greene, R. and Korn, D. (1970). J. Biol. Chem. 245, 254-261. Sedwick, W.D., Wang, T.S. and Korn, D. (1975). J. Biol. Chem. 250, 7045-7056. Richardson, J.P. (1969). Prog. Nucl. Acid Res. 9, 75-116. Hartmann, G., Honikel, K.O., Knisel, F. and Nuesch, J. (1967). Biochim. Biophys. Acta 145, 843-844. Scheffler, I.E. and Richardson, C.C. (1972). J. Biol. Chem.
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