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The sensitivity of terminal deoxynucleotidyl transferase (TdT) assay methods was examined by using a mixture of the TdT-positive lymphoblastic leukemia cell line NALM-18 and the TdT-negative erythroleukemia cell line K-562. The biochemical assay could detect TdT activity in the mixture containing NALM-18 cells at concentrations of more than 10 percent. The immunofluorescent (IF) method could detect positive cells in the mixture containing NALM-18 cells at concentrations of more than 1 percent. Furthermore, an approximately 10(5)-fold increase in sensitivity was obtained by the combination of RT-PCR and subsequent Southern blotting, as compared to biochemical assay. In many leukemia cases the expression of TdT-mRNA corresponded well to that of TdT protein. However, in some patients with leukemia, only TdT-mRNA was detectable by RT-PCR without any expression of TdT protein. A PCR-based technique enables us to detect TdT transcripts at the highest sensitivity, but does not allow the characterization of each positive cell. IF analysis is simple and sensitive, but may sometimes cause nonspecific reactions. All these techniques have some advantages and some faults, therefore, the results obtained from clinical studies using these techniques should be interpreted with caution.

Terminal deoxynucleotidyltransferase activity is absolutely dependent on the presence of a divalent cation in the reaction mixture. This requirement can be satisfied by either Mg2+, Co2+, or Mn2+. When Mg2+ is used, the reaction rate is inhibited by metal ligands, and this inhibition can be reversed by Zn2+. Reaction rates in Mg2+ are also stimulated by the addition of micromolar amounts of Zn2+. To examine the role of Zn2+ in terminal transferase catalysis we analyzed for Zn2+ in homogeneous recombinant human terminal transferase preparations and found that Zn2+ is not an intrinsic part of enzyme molecule. Analysis of Zn2+ binding to terminal transferase under equilibrium conditions shows about 0.3 g of atom of Zn2+/mol of enzyme, suggesting that Zn2+ forms an easily dissociable complex with the enzyme molecule. Kinetic analyses showed that the stimulatory effect of Zn2+ is observed in several buffer systems. Zn2+ increases the affinity of the enzyme for the initiator about 2-fold and decreases affinity for dATP more than 10-fold, resulting in an increase in the apparent Vmax of the reaction. Using a 3'-ended 2',3'-dideoxyoligonucleotide as an inhibitor demonstrates that the inhibitor has no effect on the reaction rate in the absence of Zn2+ but is competitive with respect to the initiator in the presence of Zn2+. These results suggest that Zn2+ is a positive effector for terminal transferase, interacting with oligonucleotide and enzyme near the initiator binding site. Binding of Zn2+ to the enzyme appears to induce conformational changes that greatly increase the Vmax of the reaction with a concomitant decrease in the affinity of the enzyme for dNTP.

Human terminal deoxynucleotidyltransferase was used to produce mouse monoclonal antibodies. Three different assays were used to screen for terminal transferase antibodies and antibody-producing hybridoma cells: an enzyme binding assay, an enzyme-linked immunosorbent assay, and a "terminal transferase-specific antibody" cytochemical procedure that allows visualization of the homogeneity of the hybridoma clones producing antibodies to terminal transferase. Of the 12 mouse hybridoma clones isolated, seven produce IgG1 and five produce IgG2a immunoglobulins. Only one of these monoclonal antibodies is inhibitory to human terminal transferase activity. Four of the monoclonal antibodies react with terminal transferase peptides after separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the remainder appear to recognize only native determinants on the human enzyme. Cross-reactivity studies with purified calf thymus enzyme show that although four of the monoclonal antibodies bind to native enzyme, none react with calf thymus terminal transferase peptides after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting structural differences within the cross-reactive determinants. A more general survey on the affinity of these monoclonal antibodies to human terminal transferase for the enzyme from other species using an enzyme-linked immunosorbent assay demonstrates that none of the monoclonal antibodies reacts with chicken thymus terminal transferase. The affinity of binding for terminal transferase from other mammals to this panel of monoclonal antibodies varies with the clones as expected, but few bind as strongly as the human enzyme. These results suggest that although antigenic determinants are conserved through evolution, fine structural changes within the determinants do occur.

Complementary DNA clones representing the 3' half, the 5' half, and the entire coding region of the human terminal deoxynucleotidyltransferase gene (TdT; DNA nucleotidylexotransferase, nucleosidetriphosphate: DNA deoxynucleotidylexotransferase, EC 2.7.7.31) were used to screen a panel of mouse X human somatic cell hybrid DNAs to determine the chromosomal location of the human TdT gene. The results of the Southern transfer analysis of hybrid DNAs indicate that the gene for TdT is located on human chromosome 10. The in situ hybridization technique was then used to further localize the gene for TdT to region q23-q25 of human chromosome 10.

We have established stably transformed mammalian cell lines expressing recombinant human terminal deoxynucleotidyl transferase. A 58 kDa, enzymatically active protein is produced by these cell lines. Using the lacI gene of pJYMib shuttle vector as mutagenic target, we found no increase in mutation rates in cells expressing terminal deoxynucleotidyl transferase compared to controls. Our results suggest that the presence of terminal deoxynucleotidyl transferase alone in mammalian cells does not increase mutation rates.

Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.

A high molecular weight preparation of terminal transferase containing 58,000- and 44,000-dalton peptides has been purified from calf thymus glands. The relationship of these terminal transferase peptides to the low molecular weight form was established with an immunoblot procedure using rabbit antibody directed against the homogeneous calf thymus low molecular weight terminal transferase (32,000 daltons). The 58,000- and 44,000-dalton enzyme species are each shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. These results suggest that the homogeneous terminal transferase previously described is derived from the higher molecular weight species by proteolysis during fractionation. Controlled degradation of the high molecular weight calf thymus terminal transferase with trypsin produces fully active enzyme containing alpha- and beta-peptides similar to those found in the 32,000-dalton species. Isoelectric focusing experiments show a decrease of isoelectric pH of the enzyme with proteolysis.

A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned.

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.