Terminal deoxynucleotidyl transferase enzyme

Terminal transferase Terminal deoxynucleotidyl transferase. Enzyme... 

Terminal deoxynucleotidyl transferase enzyme catalog M1871/M1872 international distributors available on web site]... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Many other enzymes can be used to remove or add groups to the ends of the DNA molecule. The three major ones are alkaline phosphatase, which removes a phosphate group from the 5 terminus polynucleotide kinase, which adds a phosphate group to a free 5 terminus and terminal deoxynucleotidyl transferase, which adds one or more deoxynucleotides to the 3 terminus. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

There is an alternative method of generating cohesive tails. The enzyme calf thymus terminal (deoxynucleotidyl) transferase adds deoxynucleoside monophosphate residues from 5 -deoxynucleoside triphosphates to protruding 3 -hydroxyl termini in the absence of a template. For example, as shown in equations 14.5 to 14.7,... 

TUNEL The TUNEL assay represents a historical low point in attempts to coin acronyms. The Terminal deoxynucleotidyl transferase mediated dC/TP Nick End-Labeling assay labels the ends of DNA with fluorescent UTP. Apoptotic cells often have fragmented DNA these fragments will provide more substrate for the enzyme terminal deoxynucleotidyl transferase. Apoptotic cells can therefore be enumerated by flow cytometry according to their increased intensity in the TUNEL assay. 

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

Both ds and. v.v DNA (e.g., oligomers) can be efficiently tailed at the 3 -end using terminal deoxynucleotidyl transferase (TdTase). This enzyme has a broad specificity (Deibel et al., 1985) but the efficiency of incorporation of the various nucleotides differs considerably. TdTase, like T4 PNKase, is quite sensitive to contaminants such as Na or protein and does not label as efficiently recessed ends as protruding ends. In case a single nucleotide is to be added, it is... 

Lymphoblastic lymphoma and acute lymphoblastic leukemia are morphologically and immunophenotypi-cally the same disease and are distinguished on clinical grounds. Although the majority of lymphoblastic leukemias are of B lineage, only approximately 20% of lymphoblastic lymphomas express B-cell markers. Practically all cases of lymphoblastic leukemia/lym-phoma produce an enzyme, terminal deoxynucleotidyl transferase (TdT), involved in gene rearrangement. TdT marks the nucleus of lymphoblasts. CD 19 is expressed in almost all lymphoblastic lymphomas but... 

Terminal deoxynucleotidyl transferase (TdT), an enzyme found in bone marrow and thymus tissue, can extend a DNA primer by 5 —> 3 polymerization using deoxyri-bonucleoside triphosphates as substrates. The primer must be at least three nucleotides... 

What is known concerning the activities of DNA polymerases isolated from various eukaryotes DNA polymerase (replicative deoxynucleotidyl transferase) has been isolated from calf thymus (Bollum, 1960) and catalyzes the addition of deoxyribonucleoside triphosphates to the 3 4iydroxyl terminus of a primer DNA in a reaction which has an absolute requirement for template DNA. In addition, terminal deoxynucleotidyl transferases (end addition enzymes) have been recognized as separate enzymes from calf thymus (Krakow et al., 1962) and physically separated from the enzyme DNA polymerase (Yoneda and Bollum, 1965). The terminal deojQ nucleotidyl transferase enzymes catalyze the incorporation of mononucleotide units from the nucleoside-5 -triphosphates into 3 -terminal positions of DNA in a reaction not template directed. The template requirements of the calf thymus polymerases have been discussed by Bollum... 

DNA pol a with primase was assayed in a reaction mixture (0.2 ml) containing 50 mM Tris-HQ buffer, pH 7.4, 10 mM MgCU, 1 mM ATP, 2.5 Mg of poly(dT), 20mM[ H]-dATP (35 cpm pmoF ) and an appropriate amount of the enzyme. The reaction was carried out at 37°C for 1 h. DNA polymerase a and i3 activity was assayed with activated DNA as template-primer. Details of the assay will be described elsewhere [30]. The assay of DNA ligase [12,13], Ca ", Mg -dependent endonuclease [14], and terminal deoxynucleotidyl transferase [15] was carried out according to the respective reported method. 

Fig. 3A-C. Inhibition of DNA ligase II, DNA polymerase jS, and terminal deoxynucleotidyl transferase by poly(ADP-ribos)ylation. Highly purified DNA ligase II (5 Mg, A), DNA polymerase ]3 (5 Mg, B), and terminal deoxynucleotidyl transferase (0.65 Mg, Q from bovine thymus were incubated in a poly(ADP-ribos)ylating reaction mixture as described in Sect. 2 except that the concentration of NAD was changed as indicated. After incubation, respective enzyme activity was assayed. The activity of a control sample incubated without NAD in each experiment was set at 100%...

Fig. 6A,B SDS-polyacrylamide gel electrophoresis of Hloligo(ADP-ribos)ylated DNA polymerase (3 and poly(ADP-ribos)ylated terminal deoxynucleotidyl transferase. [ H]oligo(ADP-ribos)ylated bovine DNA polymerase (3 (A) and poly(ADP-ribos)ylated terminal deoxynucleotidyl transferase (B) were analyzed. A Lanes 1 and 2 indicate stained protein band and the fluorography of the same gel. B Lanes 3-5 indicate protein staining of a control sample (lane 3) incubated without NAD, a poly(ADP-ribos)ylated enzyme (lane 4), and an alkaline-treated sample of poly(ADP-ribos)ylated enzyme (lane 5)...

The response of terminal deoxynucleotidyl transferase to poly(ADP-ribos)ylation in vitro was also quite similar to that of other enzymes already described. The enzyme activity showed time-dependent (data not shown) and NAD" -dependent (Fig. 3) decrease upon an incubation in the poly(ADP-ribos)ylating system. The components... 

Yoshida S, Nakamura H (1981) Terminal deoxynucleotidyl transferase in gene engineering. Protein Nucleic Acid Enzymes 26 569-574... 

Terminal deoxynucleotidyl transferase (TdT, 10-20 U/pL) can be purchased from Gibco-BRL. The enzyme is supplied with a vial of 5X reaction buffer (500 mM potassium cacodylate, pH 7 2, 10 mM C0CI2,1 mM DTT) Store at -20°C. Note that potassium cacodylate and C0CI2 are highly toxic and should be handled with care... 

Another common method for measuring apoptosis is the DNA labeling technique, terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, labeling of DNA strand breaks at the 3 end is achieved by using the enzyme, terminal deoxynucleotidyl transferase (TdT) and either a radioactive or a fluorescently-Iabeled deoxynucleotide. This method provides a very sensitive measurement of DNA strands breaks. However, as DNA strand... 

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