Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.     

The reaction of alkylating agents with cyclic 3',5'-nucleotide phosphodiesterase

The reaction of anti-tumour alkylating agents with beef heart cyclic nucleotide phosphodiesterase (adenosine 3',5'-monophosphate phosphohydrolase, EC 3.1.4.c) has been investigated. This enzyme exists in two forms differing in their Michaelis-Menten K_m values. Chlorambucil [p-(di-2-chloroethylamino)-phenyl-butyric acid] inhibits the form of the enzyme with a low K_m value with a velocity constant for inactivation three times that for inhibition of the high K_m form. While the monofunctional N-ethyl analogue of chlorambucil is ineffective as an inhibitor of either form of the enzyme, iodoacetate inhibits both forms, though the velocity constant for inactivation of each form is much less than that for chlorambucil. Also the rate of inactivation of each form does not significantly differ. A cross-linking mechanism for the inactivation of regulatory enzymes is proposed.     

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.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Organ-specific distribution of isozymes of 5'-nucleotide phosphodiesterase in mouse

1. The distribution of isozymes of 5'-nucleotide phosphodiesterase (E.C.3.1.4.1) was examined in various organs of mouse, including liver, spleen, pancreas, heart, lung, kidney, brain and blood. 2. Five isozymes were identified and designated as isozymes I through V. 3. These isozymes are distributed unevenly with respect to the various organs and clear differences were observed in the patterns of distribution among the organs examined. 4. The level of these isozymes was compared in serum of neonate and adult mice, and a higher level of isozyme I and a lower level of isozyme IV were found in neonates compared to adults. 5. These results suggest that each isozyme has different functional roles in individual organs and that these isozymes may be involved in proliferation and development of cells.     

Demonstration of promoter function for the 5'-flanking region of the human gene for terminal deoxynucleotidyl transferase

Promoter activity was detected by transfection of pBS vectors that carried the 5'-flanking region of the gene for human terminal deoxy-nucleotidyltransferase (TdT) and the entire human TdT cDNA into COS7 monkey kidney cells, with direct assay of the expressed enzymatically active TdT. The synthesis of TdT was also detected immunologically using polyclonal rabbit antibodies against bovine TdT. One fifth of the TdT activity detected in normal thymocytes was detected in transfected COS7 cells. This results suggests that a lymphoid-specific regulatory system is present in B or T-cells to promote the effective expression of TdT.     

Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.     

A new scheme for the synthesis of 5'-nucleotide phosphonate analogs

A convenient and general method is proposed for the synthesis of 5'-nucleotide phosphonate analogs starting from 5-deoxy-1,2-O-isopropylidene-alpha-D-xylo-hexofuranose.     

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.     

Intracellular localization of terminal transferase during the cell cycle.

Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.     

Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A

Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV-mediated substrate cross-linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'-5' penta- and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate- and primer-binding domains in TdT.     

Expression and processing of recombinant human terminal transferase in the baculovirus system

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.     

Effect of phospholipid vesicles on the activity of terminal transferase.

The terminal transferase activity is modified in the presence of lipid vesicles. A deep inhibitory effect takes place with phosphatidylserine and phosphatidylinositol, while some stimulation is present with sphingomyelin and almost no effect has been detected with phosphatidylethanolamine vesicles. These effects seem to be related to the charge properties of the lipid membranes.A possible involvement of phospholipids in the mechanism of action of the terminal transferase is suggested.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Estimation of the reaction efficiency in polymerase chain reaction

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.