Volume 4 Number 12 December 1977
Volume 4
Number
12
December
1977
Nucleic Acids Research
Nucleic Acids
Research
A critical comparison of commonly used procedures for the assay of terminal deoxynucleotidyl
A critical comparison of commonly used procedures for the assay of termilial deoxynucleotidyl transferase in crude tissue extracts
Mary Sue Coleman
Department of Biochemistry, University of Kentucky, Lexington, KY 40506, USA Received 16 September 1977
ABSTRACT
Terminal deoxynucleotidyl transferase (TdT) is a non-template directed DNA polymerase normally found in vertebrate thymus and bone marrow. Quantitative assay of TdT activity is being widely used as a tool in the differential diagnosis of acute leukemias in man. Clinical specimens of blood and bone marrow often contain 107 or fewer cells and require a specific and rapid assay for transferase which can be carried out in crude cell extracts. Commonly used assay methods do not meet these requirements, but can be easily modified to do so. INTRODUCTION Terminal deoxynucleotidyl transferase (TdT) activity has been associated with certain types of leukemic cells (1-5) and has been postulated to play an essential role in the differentiation of lymphoid cells (6,7). It is often desirable and necessary to assay this enzyme activity in crude extracts where low levels of enzyme are present. Since TdT is a non-template directed DNA polymerase, the most commonly used initiators are oligo or polydeoxynucleotides, or activated DNA with a single deoxynucleoside triphosphate as the monomer. In this report two problems inherent in current assay procedures are examined: (1) acid solubility of the reaction product with oligomer initiators and (2) cross reactivity of DNA polymerase owhen activated DNA is used with a single deoxynucleoside 5'-triphosphate as initiator. Modifications of these procedures are examined in order to optimize the TdT assay using substrates from commercial sources. MATERIALS AND METHODS Labeled deoxyguanosine 5'-triphosphate and thymidine 5'-triphosphate were purchased from New England Nuclear, Boston, Mass. (sp. activity 100 cpm/pmole) Unlabeled deoxynucleoside triphosphates were from Sigma (St. Louis, Mo.). Nucleoside triphosphates were further purified by chromatography on QAESephadex A-25 (Pharmacia, Uppsala) (8). Oligo d(pA)12 18 was purchased from G Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4305
Nucleic Acids Research Collaborative Research (Waltham, Mass.). Dr. F. J. Bollum generously provided poly d(pA).0 and oligo d(pA)11. Activated DNA was prepared in this laboratory. All other chemicals were reagent grade from commercial sources. Lymphoblastoid tissue culture cells used were RPMI-8402, HEM Research Associates (Rockville, Md.) and CCRF CEM, American Type Culture Collection (Rockville, Md.). The cells, grown in suspension culture, were sonicated for 20 seconds (Branson Sonifier) in 0.25 M potassium phosphate, pH 7.5, 1 mM mercaptoethanol. The sonicates were centrifuged for 60 minutes at 100,000 x g and used for enzyme assays. Enzyme assays were carried out in a total volume of 125 pl. TdT assay I contained 1 mM 3H-dGTP, 1 mM 2-mercaptoethanol, 1 mM MnCl2, 0.2 M potassium cacodylate, pH 7.5, initiator [d(pA)TO, d(pA)12-18 or d(pA) 1] and tissue extract. At timed intervals, 25 pl aliquots were either spotted onto Whatman GF/C disks and washed as has been described (9) or spotted onto Whatman DE-81 ion-exchange paper and dropped into a beaker containing 5% potassium dibasic phosphate. The DE-81 disks were batch washed three times with 5% potassium dibasic phosphate, twice with 95% ethanol and twice with ether. After drying, the disks were placed into glass scintillation vials with 1 ml of Soluene-350 (Packard Instrument Co.) and digested overnight at 37°C. Following digestion, 10 ml of Dimilume 30 (Packard) were added to each vial for counting in a Packard Liquid Scintillation Counter. A third procedure was used for some samples. Aliquots of the reaction mixture were spotted onto a strip of DE-81 chromatography paper on which unlabeled dGTP had been spotted at the origin. Strips were 23 cm wide x 10 cm long, and each strip had 12 penciled channels for product application. The strips were developed in 5% potassium dibasic phosphate by descending chromatography. The chromatography was completed in two hours. The DE-81 strips were dried at 1050C, the oligomer product at the origin was cut out, solubilized and counted as has been described. This method is advantageous because the background radioactivity is much lower than with the batch washing procedure. TdT assay II contained 0.1 mM 3H*dTTP, 1 mM 2-mercaptoethanol, 8 mM MgCl2 or 1 mM MnC12, 50 mM Tris pH 8.3, and 0.32 A260 units of activated DNA. At timed intervals, 25 ul aliquots were spotted onto Whatman GF/C disks and washed as has been described (9). The assay contained 0.1 mM 3H'dTTP, 0.1 mM dGTP, 0.1 mM dCTP, DNA polymerase 0.1 mM dATP, 1 mn 2-mercaptoethanol, 8 mM MgCl2, 50 mM Tris, pH 8.3 and 0.32 A260 units of activated DNA. In some experiments 10 mM N-ethylmaleimide was incubated with the enzyme preparation prior to the start of the enzyme assay (10, 11). -
4306
Nucleic Acids Research RESULTS Teminal transferase assays were carried out in crude extracts using d(pA)12 18, d(pA)50 and activated DNA as the initiator since all of these methods are currently being used in various laboratories to measure this enzyme activity quantitatively. Initially, it was observed that enzyme activities were much lower using d(pA)12 18 as the initiator than with d(pA)5when the products were collected on glass fiber disks and washed with 5% trichloracetic acid. In order to investigate the apparent difference in reaction rates with these initiators, saturation curves were determined (Figure 1). While the saturation curve for the d(pA)g0 closely approximates Michaelis-Minton Kinetics, the saturation curve using d(pA)12 18 is unorthodox. With this initiator, it is impossible to determine Km or Vmax values. The curve obtained suggests that at very low initiator concentrations, the enzyme is polymerizing enough monomer units to the limited number of available 3'OH ends to render the product acid insoluble. At higher initiator concentrations, where more 3'-OH groups are available, the enzyme appears to be adding only a few monomer units and the product is largely acid soluble. Therefore, with acid precipitation techniques, low levels of terminal transferase cannot be measured in crude extract using commercially available initiators. Utilization of an alternate technique for product analysis, i.e. ion exchange chromatography or ion exchange absorption and batch washing procedures circumvents the acid solubility problem ('12-14). Saturation curves determined using ion-exchange absorption of the reaction products onto DE-81 paper are shown in Figure 2. Wheni d(pA)-g-, d(pA)1218, or d(pA)11 is used, reasonable saturation curves are obtained, indicating that the reaction products are consistentiy binding to the ion-exchange paper. Km values obtained for the three initiators were 1.04 ± 0.17 pm, 1.47 ± 0.17 PM, and 1.10 ± 0.05 PM. This particular experiment utilized a batch washing technique. However, the short chromatography procedure detailed in Materials and Methods generates equally satisfactory results with very low levels of background radioactivity. Using the chromatography procedure, 100 samples can be run concurrently in a standard chromatography jar. In these experiments, 3H-dGTP was utilized as the monomer at a specific activity of 100 cpm/pmole. The use of a tritium labeled compound necessitates solubilization of the product from the ion-exchange paper. Soluene-350 was preferable to any other commercially available product since digestion time was very short and background levels of radioactivity were low. It is also possible to use a32P dGTP and omit the solublization step. Nucleotides 4307
Nucleic Acids Research
lo
W 7
7G0 4
0.1 0.2
d(pA)
12-B8
~0~ 0.5
ID aO
4.0
6.0
8.0
0
20
INITIATOR CONCENTRATION (pM)
Figure 1. Initiator Saturation Curves for the TdT Assay Using Acid Precipitability for Product Analysis. Saturation curves for d(pA)F and d(pA)12 18 were generated at a constant dGTP concentration of lm4. The reaction products were spotted on GF/C squares and batch washed as described in Methods.
labeled with 14C cannot be used in assays where low levels of terminal transferase are present since the specific activity of these compounds is not high enough. Another initiator in common use for assay of terminal transferase is activated DNA along with a single deoxynucleoside 5' triphosphate. The assumption is that DNA polymerases will not utilize activated DNA as a template under these conditions and that terminal transferase activity can be selectively measured. If the sample of interest contains very high levels of terninal transferase (>20 units/108 cells), this assertion may be valid. However, very often it is desirable to detect low levels of the enzyme activity (
Volume 4
Number
12
December
1977
Nucleic Acids Research
Nucleic Acids
Research
A critical comparison of commonly used procedures for the assay of terminal deoxynucleotidyl
A critical comparison of commonly used procedures for the assay of termilial deoxynucleotidyl transferase in crude tissue extracts
Mary Sue Coleman
Department of Biochemistry, University of Kentucky, Lexington, KY 40506, USA Received 16 September 1977
ABSTRACT
Terminal deoxynucleotidyl transferase (TdT) is a non-template directed DNA polymerase normally found in vertebrate thymus and bone marrow. Quantitative assay of TdT activity is being widely used as a tool in the differential diagnosis of acute leukemias in man. Clinical specimens of blood and bone marrow often contain 107 or fewer cells and require a specific and rapid assay for transferase which can be carried out in crude cell extracts. Commonly used assay methods do not meet these requirements, but can be easily modified to do so. INTRODUCTION Terminal deoxynucleotidyl transferase (TdT) activity has been associated with certain types of leukemic cells (1-5) and has been postulated to play an essential role in the differentiation of lymphoid cells (6,7). It is often desirable and necessary to assay this enzyme activity in crude extracts where low levels of enzyme are present. Since TdT is a non-template directed DNA polymerase, the most commonly used initiators are oligo or polydeoxynucleotides, or activated DNA with a single deoxynucleoside triphosphate as the monomer. In this report two problems inherent in current assay procedures are examined: (1) acid solubility of the reaction product with oligomer initiators and (2) cross reactivity of DNA polymerase owhen activated DNA is used with a single deoxynucleoside 5'-triphosphate as initiator. Modifications of these procedures are examined in order to optimize the TdT assay using substrates from commercial sources. MATERIALS AND METHODS Labeled deoxyguanosine 5'-triphosphate and thymidine 5'-triphosphate were purchased from New England Nuclear, Boston, Mass. (sp. activity 100 cpm/pmole) Unlabeled deoxynucleoside triphosphates were from Sigma (St. Louis, Mo.). Nucleoside triphosphates were further purified by chromatography on QAESephadex A-25 (Pharmacia, Uppsala) (8). Oligo d(pA)12 18 was purchased from G Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4305
Nucleic Acids Research Collaborative Research (Waltham, Mass.). Dr. F. J. Bollum generously provided poly d(pA).0 and oligo d(pA)11. Activated DNA was prepared in this laboratory. All other chemicals were reagent grade from commercial sources. Lymphoblastoid tissue culture cells used were RPMI-8402, HEM Research Associates (Rockville, Md.) and CCRF CEM, American Type Culture Collection (Rockville, Md.). The cells, grown in suspension culture, were sonicated for 20 seconds (Branson Sonifier) in 0.25 M potassium phosphate, pH 7.5, 1 mM mercaptoethanol. The sonicates were centrifuged for 60 minutes at 100,000 x g and used for enzyme assays. Enzyme assays were carried out in a total volume of 125 pl. TdT assay I contained 1 mM 3H-dGTP, 1 mM 2-mercaptoethanol, 1 mM MnCl2, 0.2 M potassium cacodylate, pH 7.5, initiator [d(pA)TO, d(pA)12-18 or d(pA) 1] and tissue extract. At timed intervals, 25 pl aliquots were either spotted onto Whatman GF/C disks and washed as has been described (9) or spotted onto Whatman DE-81 ion-exchange paper and dropped into a beaker containing 5% potassium dibasic phosphate. The DE-81 disks were batch washed three times with 5% potassium dibasic phosphate, twice with 95% ethanol and twice with ether. After drying, the disks were placed into glass scintillation vials with 1 ml of Soluene-350 (Packard Instrument Co.) and digested overnight at 37°C. Following digestion, 10 ml of Dimilume 30 (Packard) were added to each vial for counting in a Packard Liquid Scintillation Counter. A third procedure was used for some samples. Aliquots of the reaction mixture were spotted onto a strip of DE-81 chromatography paper on which unlabeled dGTP had been spotted at the origin. Strips were 23 cm wide x 10 cm long, and each strip had 12 penciled channels for product application. The strips were developed in 5% potassium dibasic phosphate by descending chromatography. The chromatography was completed in two hours. The DE-81 strips were dried at 1050C, the oligomer product at the origin was cut out, solubilized and counted as has been described. This method is advantageous because the background radioactivity is much lower than with the batch washing procedure. TdT assay II contained 0.1 mM 3H*dTTP, 1 mM 2-mercaptoethanol, 8 mM MgCl2 or 1 mM MnC12, 50 mM Tris pH 8.3, and 0.32 A260 units of activated DNA. At timed intervals, 25 ul aliquots were spotted onto Whatman GF/C disks and washed as has been described (9). The assay contained 0.1 mM 3H'dTTP, 0.1 mM dGTP, 0.1 mM dCTP, DNA polymerase 0.1 mM dATP, 1 mn 2-mercaptoethanol, 8 mM MgCl2, 50 mM Tris, pH 8.3 and 0.32 A260 units of activated DNA. In some experiments 10 mM N-ethylmaleimide was incubated with the enzyme preparation prior to the start of the enzyme assay (10, 11). -
4306
Nucleic Acids Research RESULTS Teminal transferase assays were carried out in crude extracts using d(pA)12 18, d(pA)50 and activated DNA as the initiator since all of these methods are currently being used in various laboratories to measure this enzyme activity quantitatively. Initially, it was observed that enzyme activities were much lower using d(pA)12 18 as the initiator than with d(pA)5when the products were collected on glass fiber disks and washed with 5% trichloracetic acid. In order to investigate the apparent difference in reaction rates with these initiators, saturation curves were determined (Figure 1). While the saturation curve for the d(pA)g0 closely approximates Michaelis-Minton Kinetics, the saturation curve using d(pA)12 18 is unorthodox. With this initiator, it is impossible to determine Km or Vmax values. The curve obtained suggests that at very low initiator concentrations, the enzyme is polymerizing enough monomer units to the limited number of available 3'OH ends to render the product acid insoluble. At higher initiator concentrations, where more 3'-OH groups are available, the enzyme appears to be adding only a few monomer units and the product is largely acid soluble. Therefore, with acid precipitation techniques, low levels of terminal transferase cannot be measured in crude extract using commercially available initiators. Utilization of an alternate technique for product analysis, i.e. ion exchange chromatography or ion exchange absorption and batch washing procedures circumvents the acid solubility problem ('12-14). Saturation curves determined using ion-exchange absorption of the reaction products onto DE-81 paper are shown in Figure 2. Wheni d(pA)-g-, d(pA)1218, or d(pA)11 is used, reasonable saturation curves are obtained, indicating that the reaction products are consistentiy binding to the ion-exchange paper. Km values obtained for the three initiators were 1.04 ± 0.17 pm, 1.47 ± 0.17 PM, and 1.10 ± 0.05 PM. This particular experiment utilized a batch washing technique. However, the short chromatography procedure detailed in Materials and Methods generates equally satisfactory results with very low levels of background radioactivity. Using the chromatography procedure, 100 samples can be run concurrently in a standard chromatography jar. In these experiments, 3H-dGTP was utilized as the monomer at a specific activity of 100 cpm/pmole. The use of a tritium labeled compound necessitates solubilization of the product from the ion-exchange paper. Soluene-350 was preferable to any other commercially available product since digestion time was very short and background levels of radioactivity were low. It is also possible to use a32P dGTP and omit the solublization step. Nucleotides 4307
Nucleic Acids Research
lo
W 7
7G0 4
0.1 0.2
d(pA)
12-B8
~0~ 0.5
ID aO
4.0
6.0
8.0
0
20
INITIATOR CONCENTRATION (pM)
Figure 1. Initiator Saturation Curves for the TdT Assay Using Acid Precipitability for Product Analysis. Saturation curves for d(pA)F and d(pA)12 18 were generated at a constant dGTP concentration of lm4. The reaction products were spotted on GF/C squares and batch washed as described in Methods.
labeled with 14C cannot be used in assays where low levels of terminal transferase are present since the specific activity of these compounds is not high enough. Another initiator in common use for assay of terminal transferase is activated DNA along with a single deoxynucleoside 5' triphosphate. The assumption is that DNA polymerases will not utilize activated DNA as a template under these conditions and that terminal transferase activity can be selectively measured. If the sample of interest contains very high levels of terninal transferase (>20 units/108 cells), this assertion may be valid. However, very often it is desirable to detect low levels of the enzyme activity (
