Expression of human terminal deoxynucleotidyl transferase in Escherichia coli

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.     

Purification of a high molecular weight human terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyltransferase has been purified from lymphoblasts of leukemic patients. The enzyme has a molecular weight of approximately 62,000 as determined by gel filtration and nondenaturing gel electrophoresis and is not dissociated into subunits by sodium dodecyl sulfate. In contrast, the terminal transferase enzyme from calf thymus has a molecular weight of 42,000 as determined by gel filtration, and is dissociated into 2 subunits of Mr 30,000 and 8,000 by sodium dodecyl sulfate. The enzyme has an isoelectric point of 8.2 and kinetic characteristics which are similar to those of calf thymus terminal transferase. The apparent Km for purine nucleotide polymerization at saturating initiator concentration with Mg2+ is 0.2 mM and with Mn2+ is 0.05 mM. Like calf terminal transferase, the reaction velocity is higher in the presence of Mg2+ than Mn2+. ATP inhibits the reaction catalyzed by terminal transferase isolated from human lymphoblasts due to mutual recognition of ATP and dATP by a common site on the enzyme. Preliminary experiments indicate that human terminal transferase may contain a small amount of carbohydrate. This report represents the first purification to near homogeneity of terminal transferase from a tissue source other than calf thymus.     

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.     

Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aureus V-8 protease results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, β, is modified from a molecular weight of 30,500 to 25,500. The small subunit, α, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from pI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further by novel methods involving limited proteolysis without concomitant loss in enzymatic function.     

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.     

Terminal deoxynucleotidyl transferase in human brain

A DNA polymerase lacking template direction for base selection has been partially purified from human brain. The molecular size, optimum reaction conditions, initiator preferences and chemical inhibitors of the brain enzyme were similar to calf thymus terminal deoxynucleotidyl transferase (TdT). TdT has been found only in the thymus, and now in brain. The possibility exists that its function is related to biological property unique to these two organs.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

Developmental changes in terminal deoxynucleotidyl transferase of the chicken thymus

Terminal deoxynucleotidyl transferase activity begins to be detectable in the thymus of 14-day old chicken embryos. It reaches a maximum 3 weeks after hatching, and persits at a fairly high level in 21-weeks old chickens. Multiple chromatographic forms of TdT are detected, and the relative proportions of these forms change during the development of the chicken.     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase

Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The probes were then detected with either streptavidin-alkaline phosphatase complex or anti-fluorescein antibodies and alkaline phosphatase-conjugated secondary antibodies. Tailing conditions were optimized and the probes were tested for detection of Escherichia coli ST1a enterotoxin DNA and rotavirus RNA.     

Poly(ADP-ribosyl)ation of terminal deoxynucleotidyl transferase in vitro

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP-ribose)-synthesizing system containing purified bovine thymus poly(ADP-ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly(ADP-ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose-dependent manner, suggesting that the transferase is functioning as an acceptor for ADP-ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo (ADP-ribosyl)ated. When the product was further incubated in the Mg2+-fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP-ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP-ribose) and the polymer bound to poly(ADP-ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP-ribosyl)ation of the transferase itself.