Terminal Deoxynucleotidyl Transferase Activity Research Articles

Gene Expression of Terminal Deoxynucleotidyl Transferase in Neoplastic Cells of Leukemia and Lymphoma

Expression levels of terminal deoxynucleotidyl transferase (TdT) mRNA in fresh leukemia and lymphoma cells were measured by northern blotting analysis. Bands of 2.1 kb mRNA were detected in all of eight cases of TdT activity‐positive leukemias: two cases of null‐cell acute lymphoblastic leukemia (null‐ALL), two of common ALL, one of pre‐B ALL, one of T‐ALL, and two of chronic myelogenous leukemia in blastic crisis. One of the null‐ALL and one of the common ALL cases also showed large TdT mRNA (3.3 kb). Since all TdT activity‐positive samples exhibited TdT mRNA, the TdT gene might be mainly regulated at the transcription level in leukemic cells. An elevated level of 2.1 kb TdT mRNA was also detected in one lymphoma case, where neither TdT activity nor immunoreactive TdT was detected. The extensive chromosomal abnormality demonstrated in this case might be associated with the translational anomaly of TdT.

Lymphoid V(D)J recombination: nucleotide insertion at signal joints as well as coding joints.

The coding regions of antigen receptor genes assembled by variable-diversity-joining region [V(D)J] recombination are known in many cases to have undergone deletions of several nucleotides and also to contain insertions of noncoded nucleotides at the recombined junction (the coding joint). By using extrachromosomal recombination substrates to transfect lymphoid cell lines, we show that the signal joint (the fusion of the corresponding recognition signal sequences) can also contain insertions; however, nucleotide loss from the signals is very rare. The frequency of nucleotide addition varies among pre-B-cell lines in a manner proportional to their content of terminal deoxynucleotidyltransferase. We also find frequent nucleotide additions (and deletions) at coding joints, but in this case there is no strong correlation with the level of terminal deoxynucleotidyltransferase activity. Inserts at both signal and coding joints are rich in G + C, consistent with the base utilization preference of this enzyme.

T Lymphocyte Development in Horses I. Characterization of Monoclonal Antibodies Identifying Three Stages of T Lymphocyte Differentiation

Six monoclonal antibodies reacting with equine T lymphocytes at different stages of maturation were selected from antibodies produced against lymphoid cell preparations. EqT12 and EqT13 antibodies identified subsets of cortical thymocytes with high terminal deoxynucleotidyltransferase (TdT) activity and no phytolectin responsiveness. EqT12+ thymocytes were scattered throughout the cortex while EqT13+ thymocytes were located in the subcapsular cortex. EqT12 bound to small numbers of bone marrow cells, splenocytes, and circulating lymphoid cells, but not to mature T lymphocytes. EqT13 bound to very small numbers of bone marrow cells but not to more mature lymphocytes. EqT6 and EqT7 reacted with a large population of cortical thymocytes with high TdT activity and no phytolectin responsiveness. EqT2 and EqT3 bound primarily to medullary thymocytes with low TdT activity. Eq2+ thymocytes responded to phytolectin stimulation while EqT3+ thymocytes did not. EqT2 and EqT3 bound to 33% and 91% of circulating T lymphocytes, respectively. The T lymphocytes bound by both antibodies included cells capable of suppressing a mixed lymphocyte reaction. Thus, EqT12 and EqT13 identify cells with the functional characteristics of prothymocytes. EqT6 and EqT7 identify resident cortical thymocytes, and EqT2 and EqT3 identify a subpopulation of mature T lymphocytes and all mature T lymphocytes, respectively.

Characterisation of blast cells during blastic phase of chronic myeloid leukaemia by immunophenotyping—Experience in 60 patients

The blast cell population of 60 patients with chronic myeloid leukaemia in blast crisis (CML-BC) were analyzed with a panel of monoclonal antibodies to determine the cell surface antigen phenotypes. In addition, cytochemical stains periodic acid Schiff (PAS), myeloperoxidase (MP), Sudan black B (SBB) and terminal deoxynucleotidyl transferase (TdT) were also utilized for subtyping. Nineteen cases (31.6%) expressed lymphoid phenotypes characteristic of common ALL cells and one case with extramedullary lymph node crisis expressed T-cell surface phenotypes. Thirty cases (50%) expressed solely myelomonocytic surface antigens with significant TdT activity in three. Cytochemical stains contributed to recognize only 57% of these myeloid blasts. Seven cases (11.7%) were with a mixture of heterogenous group of cells expressing phenotypic characteristics for various haemopoietic cells of different lineage—five of them from the cells of non-lymphoid series (myelomono-erythromegakaryocytic series) and the other two with cells from both lymphoid and myeloid series. Additionally, in two cases (3.3%), the precursor cells reacted only with the erythroid monoclonals. Finally, in one case, the blast cells remained unclassified due to nonreactivity with any of the monoclonals used but expressed significant TdT positivity. The response to uniform vincristine and prednisolone (V + P) therapy has shown that lymphoid blast crisis cases were highly responsive in contrast to the cases with non-lymphoid blast crisis (complete remission rate 86 vs 21.4%). The results confirm the evidence of multilineage blast crisis involving either single or mixed haemopoietic differentiation pathway and the utility of having phenotypic characterisation for designing protocols for chemotherapy in the CML patients at the time of blast crisis.

Site-selective cAMP analogs at micromolar concentrations induce growth arrest and differentiation of acute promyelocytic, chronic myelocytic, and acute lymphocytic human leukemia cell lines

Cyclic AMP (cAMP)-dependent protein kinase may play a role in the functional and morphological differentiation of leukemic cells. In this study, we showed that the cAMP analogs, potent activators of protein kinase recently shown to be selective for either site 1 or site 2 cAMP binding sites of protein kinase, demonstrate potent growth inhibition of acute promyelocytic, chronic myelocytic, and acute lymphocytic leukemic cell lines with no sign of toxicity. The growth inhibition accompanied monocytic differentiation in HL-60 cells and a loss of nuclear terminal deoxynucleotidyl transferase activity in Molt-4 leukemic cells. The growth inhibition also paralleled a decrease in c- myc protein and RI cAMP receptor protein. Thus, cAMP analogs selective for either site 1 or site 2 of the protein kinase appear to restore a coupling of proliferation and maturation in leukemic cells.

Site-Selective cAMP Analogs at Micromolar Concentrations Induce Growth Arrest and Differentiation of Acute Promyelocytic, Chronic Myelocytic, and Acute Lymphocytic Human Leukemia Cell Lines

Cyclic AMP (cAMP)-dependent protein kinase may play a role in the functional and morphological differentiation of leukemic cells. In this study, we showed that the cAMP analogs, potent activators of protein kinase recently shown to be selective for either site 1 or site 2 cAMP binding sites of protein kinase, demonstrate potent growth inhibition of acute promyelocytic, chronic myelocytic, and acute lymphocytic leukemic cell lines with no sign of toxicity. The growth inhibition accompanied monocytic differentiation in HL-60 cells and a loss of nuclear terminal deoxynucleotidyl transferase activity in Molt-4 leukemic cells. The growth inhibition also paralleled a decrease in c-myc protein and RI cAMP receptor protein. Thus, cAMP analogs selective for either site 1 or site 2 of the protein kinase appear to restore a coupling of proliferation and maturation in leukemic cells.

Monoclonal Antibodies that Recognize a Broad Range of Mammalian Terminal Deoxynucleotidyl Transferases

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for lymphocyte precursors in the bone marrow and thymus and for lymphoblastic leukemia and lymphoma cells. To simplify and enhance the detection and phenotypic analysis of these cells, we sought to develop monoclonal antibodies to this enzyme. In order to obtain antibodies that bind a variety of mammalian TdTs, mice were immunized with bovine TdT and the hybridoma secretions were screened by immunofluorescence assays on cultured TdT-positive and negative human lymphoblasts. Four monoclonal antibodies which bound specifically to TdT-positive lymphoblasts were characterized in detail. All four antibodies immunoprecipitated the native 60 kd TdT molecule from extracts of TdT-positive human lymphoblasts and bound specifically in immunoblot assays to the 43.8 and 11 kd proteolytic fragments of bovine thymus TdT. To assess whether the antibodies bound to related or distinct epitopes on bovine TdT, we measured the displacement of radiolabeled antibody from the immobilized enzyme by an excess of unlabeled heterologous antibody. These studies revealed that three of the antibodies competed for the same determinant on bovine TdT, while one antibody reacted with a distinct epitope. Antibody binding to either epitope, however, partially inhibited the enzymatic activity of bovine TdT. Specificity for TdT was tested by immunofluorescence and competition radioimmunoassays. In these assays, the antibodies did not stain a variety of known TdT-negative human hematopoietic cells and cell lines. both normal and neoplastic, nor were the antibodies displaced from purified bovine TdT by extracts of these TdT-negative cells. These results confirmed the cross-reactivity of the antibodies with human and bovine TdT. To assess cross-reactivity with TdT from other species, extracts of rabbit, mouse, and rat thymus were prepared and shown to specifically displace the antibodies from bovine TdT. Thus, these antibodies bound to TdT derived from at least five mammalian species. To determine whether these antibodies could be used to detect small subpopulations of TdT-positive cells, mixtures of TdT-positive and negative cells were prepared and stained with fluorescein conjugates of the antibodies. When assayed by flow cytometry, a population of 1% TdT-positive cells was easily detectable. We conclude that these monoclonal antibodies should be useful for the enumeration and analysis of TdT-positive cells in normal and neoplastic hematopoietic tissues from several mammalian sources, including man.

Multiparameter characterization of L3 leukemia cell populations

Multiparameter analysis of 14 consecutive patients with L3 cell populations (FAB classification) in bone marrow showed consistently high mean cellular RNA content but heterogeneity with respect to percent cells in S- and G2M-phases of the cell cycle, cell surface phenotype, cytogenetic abnormalities, cellular DNA content, and nuclear activities of terminal deoxynucleotidyl transferase. Five of the 14 patients had the characteristic profile of L3 leukemia with high RNA content, a typical t(8;14) or t(8;22) translocation, low Tdt, B-cell surface phenotype, pseudodiploid modal number of chromosomes, high proliferation and normal DNA index. The remaining 9 patients had features thought to be unusual and they were very heterogeneous. Median survival time from time of diagnosis was 115 days with no patient alive after 474 days. The survival times exhibited excellent correlation with a parametric model assuming exponential distribution. Accordingly, the presence of abnormal DI predicted for decreased, and the presence of t(8;14) or pseudodiploidy was associated with increased survival rates. However, due to the small number of patients in this study, generally applicable conclusions should be drawn only from a collection of larger numbers of cases of this rare type of leukemia.

Malignant clonal expansion of large granular lymphocytes with a Leu- 11+, Leu-7- surface phenotype: in vitro responsiveness of malignant cells to recombinant human interleukin 2

A 14-year-old Japanese female with neutropenia showed malignant proliferation of the large granular lymphocytes (LGLs). These LGLs were E rosette+ and Fc(IgG) receptor+ and therefore are referred to as T gamma lymphocytes. They were also Leu-11+ and OKT11+; however, they were clearly negative for Leu-7, OKT3, OKT8, OKM1, and HNK-1 antigens as well as for terminal deoxynucleotidyl transferase activity. Karyotype analysis revealed 47, XXX. The LGLs showed no rearrangement of T cell receptor C beta genes. The natural killer (NK) cell activity against K562 target cells was low, but was significantly augmented after stimulation by recombinant human interleukin 2 (IL 2) in contrast to minimal NK boosting by recombinant human gamma-interferon (gamma-IFN). Such a unique responsive ability to lymphokines was quite similar to that noted in fetal and cord blood cells. These LGLs also demonstrated a considerable increase in antibody-dependent cell-mediated cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activity after a short incubation with IL 2. Although in a resting stage they showed no IL 2 receptor expression as examined by anti-Tac antibody, Tac antigen appeared after IL 2 treatment followed by a marked increase in 3H-thymidine incorporation and a remarkable production of gamma-IFN. To investigate the mechanism of neutropenia, in vitro IL 2-stimulated coculture studies of these cells with normal bone marrow cells were performed. Colony formation of myeloid progenitors (CFU-C) was significantly suppressed. In addition, the conditioned medium from IL 2-stimulated LGLs indicated a remarkable suppression of CFU-C. These results suggest that these LGLs with a Leu-11+, Leu-7- surface phenotype might belong to a unique subset of pre-NK cells that are functionally and phenotypically similar to those represented at any early stage of human ontogeny and that they strongly express Tac antigen under the influence of IL 2 administration, followed by remarkable cell proliferation and gamma-IFN production.

Malignant Clonal Expansion of Large Granular Lymphocytes With a Leu-11 +, Leu-7– Surface Phenotype: In Vitro Responsiveness of Malignant Cells to Recombinant Human Interleukin 2

Abstract A 14-year-old Japanese female with neutropenia showed malignant proliferation of the large granular lymphocytes (LGLs). These LGLs were E rosette+ and Fc(IgG) receptor+ and therefore are referred to as T gamma lymphocytes. They were also Leu-11+ and OKT11+; however, they were clearly negative for Leu-7, OKT3, OKT8, OKM1, and HNK-1 antigens as well as for terminal deoxynucleotidyl transferase activity. Karyotype analysis revealed 47, XXX. The LGLs showed no rearrangement of T cell receptor C beta genes. The natural killer (NK) cell activity against K562 target cells was low, but was significantly augmented after stimulation by recombinant human interleukin 2 (IL 2) in contrast to minimal NK boosting by recombinant human gamma-interferon (gamma- IFN). Such a unique responsive ability to lymphokines was quite similar to that noted in fetal and cord blood cells. These LGLs also demonstrated a considerable increase in antibody-dependent cell- mediated cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activity after a short incubation with IL 2. Although in a resting stage they showed no IL 2 receptor expression as examined by anti-Tac antibody, Tac antigen appeared after IL 2 treatment followed by a marked increase in 3H-thymidine incorporation and a remarkable production of gamma-IFN. To investigate the mechanism of neutropenia, in vitro IL 2-stimulated coculture studies of these cells with normal bone marrow cells were performed. Colony formation of myeloid progenitors (CFU-C) was significantly suppressed. In addition, the conditioned medium from IL 2-stimulated LGLs indicated a remarkable suppression of CFU-C. These results suggest that these LGLs with a Leu- 11+, Leu-7- surface phenotype might belong to a unique subset of pre-NK cells that are functionally and phenotypically similar to those represented at any early stage of human ontogeny and that they strongly express Tac antigen under the influence of IL 2 administration, followed by remarkable cell proliferation and gamma-IFN production.

Ultramicromethod for measuring the activity of terminal deoxynucleotidyl transferase.

We describe an ultramicromethod for measurement of terminal deoxynucleotidyl transferase activity. The materials used are as proposed by Hösli (Clin Chem 1977;23:1476-84) for antenatal diagnosis. The technique is based on the methods of Beutler and Kuhl (Am J Clin Pathol 1978;70:733-7) and Yasmineh et al. (Clin Chem 1980; 26:891-5). The procedure makes it possible to reduce the blood sample to 2 mL at the most and to reduce the cost of the measurement very considerably. The technique gives results similar to those given by the conventional techniques.

Prognostic significance of terminal deoxynucleotidyl transferase activity in acute nonlymphoblastic leukemia.

Bone marrow and/or peripheral blood samples from 133 (75%) of a total of 177 consecutive previously untreated protocol patients with acute nonlymphoblastic leukemia (ANLL) were analyzed for terminal deoxynucleotidyl transferase (TdT) activity at the time of presentation. Twenty-nine (22%) were found to exhibit TdT activity (greater than or equal to 0.10 U/10(8) cells, TdT+) as measured in a biochemical microassay. There were no differences between TdT+ as compared with TdT-negative (TdT-) patients with respect to age, sex, French-American-British (FAB) classification, or the presence of Auer's rods. Remission induction rates were higher for the TdT- patients, with 68% v 48% for the TdT+ patients (P = .05). TdT- patients also experienced longer remissions (P = .003) than TdT+ patients, especially in the Auer's rod-positive subgroup (P = .002). None of five patients with TdT+ ANLL treated with vincristine and prednisone as initial therapy achieved complete remission; all required induction regimens containing daunorubicin or amsacrine in combination with cytosine arabinoside and 6-thioguanine. It is concluded that TdT activity in ANLL indicates biphenotypia or lineage infidelity and is associated with a poor prognosis on chemotherapy protocols currently used for the treatment of ANLL.

Non-germ-line elements (NGE) are present in the T cell receptor beta-chain genes isolated from the mutant mouse, motheaten (me/me).

Extra nucleotides (which we call NGE, for non-germ-line elements) are inserted at the junctions of rearranged V, D, and J segments in the immunoglobulin heavy and T cell receptor beta-chain V region genes. NGE addition helps diversity HV3 regions of these genes. It is believed that NGE are added enzymatically and without template during the joining process. Terminal deoxynucleotidyl transferase (TdT) is thought to be the cause of NGE formation. TdT is normally detected in murine thymus and bone marrow cells, but its presence in the immunodeficient mutant mouse, Motheaten (me/me), is extremely reduced in these tissues. To determine whether this TdT deficiency could affect NGE formation during the V-D-J joining of antigen receptor genes, we cloned several rearranged T cell receptor beta-chain genes from thymocytes of me/me mice. Our sequence analysis revealed that Motheaten thymus beta-chain genes have approximately 4 base pair NGE, which are comparable in size to the NGE of wild-type genes. These results do not support the idea that TdT is the NGE-forming enzyme, although it is still possible that a low but residual level of TdT is capable of NGE formation in Motheaten. Alternatively, our data may suggest that the TdT activity in Motheaten T cells is normal; however, the number of TdT+ cells is greatly reduced in the Motheaten thymus, due to a severe defect in T cell development.

Mitogen-induced changes in sialyltransferase activity in human normal and leukemic lymphocytes.

Phytohemagglutinin-P (PHA-P) and concanavalin A (Con A) induced an increase in sialyltransferase (S-T) activity of the blood lymphocytes within 24 hours. PHA-P induced the maximal increase in S-T activity in the lymphocytes at 10 micrograms/ml and with Con A at 5 micrograms/ml. PHA-P induced a greater response than that by Con A. An additional effect of these mitogens was not observed. The exposure of cell extracts to these mitogens did not induce the increase in S-T activity. PHA-P or Con A had no effect on the induction of terminal deoxynucleotidyl transferase (TdT) activity, TdT-positive cells or peanut agglutinin (PNA)-positive cells from blood lymphocytes. Therefore, the change of the lymphocytes induced by these mitogens does not indicate a reversion to more immature state of lymphocyte differentiation, but represents a process of the functional differentiation to more activated lymphocytes. These mitogens had only a slight effect on S-T activity of human T-lymphoid leukemia cell lines. The decreased effects of the mitogens on S-T activity of leukemic cells may reflect the change of the membranes due to malignant transformation or the incomplete membrane structure in undifferentiated leukemic cells.

BALB/c and CBA mice differ in the subtype of T cell involved in moloney murine leukemia virus-induced lymphomas

Subsets of T cells can be recognized by presence or absence of terminal deoxynucleotidyl transferase (TdT) or 20α-hydroxysteriod dehydrogenase (20αSDH) activity. By use of these enzyme markers Moloney murine leukemia virus (M-MuLV)-induced lymphomas were shown to involve different T-cell subtypes. In the present paper we show that the genotype of the mouse has a strong influence on the subtype of T cell involved in lymphoma. BALB/c mice preferentially develop 20αSDH-positive lymphomas, whereas CBA lymphomas often have the TdT phenotype. Comparison of the 20αSDH activity of normal bone marrow cells showed that BALB/c mice have higher enzyme levels than CBA mice. The relative availability of a certain type of cell at a critical early step in leukemogenesis may thus influence lymphoma type.

Adenosine deaminase and terminal deoxynucleotidyl transferase in human lymphomas: an aid to the diagnosis and subclassification of lymphoblastic lymphomas.

Adenosine deaminase (ADA) and terminal deoxynucleotidyl transferase (TdT) activities were determined on 97 biopsy specimens obtained from patients with non-neoplastic diseases (12 cases), Hodgkin (30 cases), and non-Hodgkin lymphomas (55 cases). Thirty additional cases were tested only for TdT. TdT was positive in 10 out of 13 lymphoblastic lymphomas (LL) examined and negative in all the other specimens, including the ten cases of the immunoblastic type. Levels of ADA above 350 U/mg of protein were found in 10 out of 12 LL tested, but not in any other specimen. The 3 TdT- LL had high contents of ADA. Therefore, all LL can be detected using both ADA and TdT markers. The 3 TdT- LL had a heterogeneous phenotype and their possible origin is discussed in view of the possibility that they constitute a rare entity distinct from the more common TdT+ LL. Very low levels of ADA (below 100 U/mg of protein) were found in chronic lymphocytic leukemia and immunocytoma, and in Burkitt's lymphoma. In other B-cell non-Hodgkin lymphomas, intermediate values between 100 and 350 U were often found, and this finding could be relevant to the different cellular origin of the various B-cell neoplasias. We conclude that ADA distribution is solid lymphoid tumors reflects the cellular origin of these neoplasias. Adenosine deaminase alone and in combination with TdT can be useful in the diagnosis and classification of childhood lymphomas in which the immature hystotypes predominate.

EFFECT OF DIETARY NUCLEOTIDES ON LYMPHOCYTE MATURATION: 175

We have shown previously that removal of purines and pyrimidines from the diet (nucleotide free diet-NFD) decreases immune responses both in vivo and in vitro. Allograft rejection and mixed lymphocyte responses and resistance to infectious organisms were all significantly influenced by NFD as compared to chow or NFD with RNA or isolated bases added. NFD affects T-helper cell number and functions as well as suppressor cell activity. In addition, NFD significantly decreases graft-versushost disease mortality in allogeneic bone marrow irradiation chimeras suggesting that NFD impairs the functional T-cell maturation. To determine if this immunologic depression in our various diet fed mice is due to impaired maturation of T-lymphocytes, we assessed lymphoid tissues for the presence of terminal deoxynucleotidyl transferase (TDT). This enzyme is a well characterized marker for immature T-lymphocytes. A statistically higher number of cells positive for TDT activity were found in the thymus and bone marrow of mice on NFD (p<0.02) as compared to cells from animals on chow or NFD with added RNA, uracil, or adenine. These results are consistent with a requirement for either a pyrimidine or purine or both for development of T-helper cell populations. These effects are likely due to a greater dependence of these cells on nucleotide salvage in Gl phase than other lymphocytes. (Supported by Grants 14030 and 35492 from the NCI.)

Thymus cell migration in a prostaglandin-mediated system

Prostaglandin (PG)-mediated T cell traffic and the nature of these emigrant T cells were analyzed by using a fluorescent activated cell sorter. Administration of indomethacin (INDO), an inhibitor of PG synthesis, increased the number of splenic T cells in normal mice but not inadult-thymectomized mice. An increase in thymus cell migrants in peripheral blood lymphocyte and splenic cell populations of mice pretreated with INDO were detected, using the method of in situ labelling of thymocytes with fluorescein diacetate. These results indicate that the increase in the T cell population in the spleen by INDO treatment results from the increase in thymus cell migration to the spleen. Such recent emigrants in the spleen were thought to have been derived from the thymus cortex, judging from the response to phytohemagglutinin and intracellular terminal deoxynucleotidyl transferase activity; however, they expressed a Thy-1 level similar to that found on peripheral T cells. These results suggested that a cortical thymocyte population was recruited from the thymus to the spleen by a PG-mediated system, but its Thy-1 level rapidly changed to that found on the peripheral T cell population.

Terminal deoxynucleotidyl transferase activity in childhood leukemia.

The activity of terminal deoxynucleotidyl transferase (Tdt) was measured in mononuclear cells of 70 children with acute leukemia, in whom raised levels of Tdt were noted at diagnosis, relapse, or during remission. Serial measurements of Tdt activity were related to the percentage of blasts in the bone marrow and the clinical course of children both during therapy and after its completion. The Tdt values did not predict relapse in children with acute lymphoblastic leukemia nor did the quantitative determination correlate with the percentage of blasts in the bone marrow.

Fluorimetric assay for terminal deoxynucleotidyl transferase activity

A fluorimetric assay for measuring terminal deoxynucleotidyl transferase activity in purified and crude enzyme preparations has been developed. Etheno-substituted deoxynucleotides are shown to be substrates of the enzyme. The assay involves polymerization of the fluorescent nucleotide 1, N 6-ethenodeoxyadenosine triphosphate (ϵdATP) on an oligodeoxynucleotide initiator, [poly(deoxyadenylic acid) with an average chain length of 50 residues] under the reaction conditions used in the standard radiometric assay. The incorporation of ϵdATP into polymer is quantitated by fluorescence after isolation and nuclease digestion of the product. The enzymological properties of etheno substrates were also determined. ϵdATP binds about twofold tighter than dATP to terminal transferase, but has a twofold-lower catalytic rate. However etheno substitution does not affect initiator binding. The fluorimetric assay is suitable for clinical analysis of terminal transferase in human leukemias, and may be a useful adjunct to recently developed immunochemical methods which detect protein, not activity.