Immunocytochemical study of recombinant terminal deoxynucleotidyl transferase (TdT) synthesized by baculovirus-infected insect cells.

The ability to overproduce terminal transferase through recombinant DNA technology should provide alternate means for generating sufficient quantities for structural and mechanistic study of this creative DNA polymerase. In this work we have investigated, at electron microscope level, the morphological modification and ultrastructural localization of synthesized human terminal transferase occurring in Sf-9 cells during recombinant baculovirus infection time. The results obtained showed that TdT is localized and stored only at the cytoplasmic level; the nucleus did not show any specific site able to link the neosynthesized TdT. The amount of the enzyme, estimate by immunostaining analysis, increased with the viral infection time. Morphological changes occurring during viral infection consist mainly of variations of cellular surface, different size and shape of cytoplasmic organelles and modification of nuclear components.     

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis

Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.     

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis

Summary Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.     

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.     

Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.     

A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis

Human DNA polymerase mu (Polμ) is a family X member that has terminal transferase activity but, in spite of a non-orthodox selection of the template information, displays its maximal catalytic efficiency in DNA-templated reactions. As terminal deoxynucleotidyl transferase (TdT), Polμ has a specific loop (loop1) that could provide this enzyme with its terminal transferase activity. When loop1 was deleted, human Polμ lacked TdT activity but improved DNA-binding and DNA template-dependent polymerization. Interestingly, when loop1 from TdT was inserted in Polμ (substituting its cognate loop1), the resulting chimaera displayed TdT activity, preferentially inserting dGTP residues, but had a strongly reduced template-dependent polymerization activity. Therefore, a specialized loop in Polμ, that could adopt alternative conformations, appears to provide this enzyme with a dual capacity: (i) template independency to create new DNA information, in which loop1 would have an active role by acting as a ‘pseudotemplate’; (ii) template-dependent polymerization, in which loop1 must allow binding of the template strand. Recent in vivo and in vitro data suggest that such a dual capacity could be advantageous to resolve microhomology-mediated end-joining reactions.     

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 (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

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.     

全染料染色法测定末端脱氧核苷酰转移酶活力

介绍一种末端脱氧核苷酰转移酶活力测定的新方法.利用全染料(stains－all)对单链寡聚核苷酸片段灵敏度较高的特点,建立了电泳─全染料染色法测定TdT酶活力,TdT酶促加长的寡聚核苷酸片段样本,经聚丙烯酰胺凝胶电泳分离、全染料染色后即可根据寡聚核苷酸片段的泳动距离推算TdT酶活力.此法所用试剂便宜,操作简便,无需大型仪器及灵敏度较高,适用于临床上白血病患者白细胞中TdT酶活力的半定量检测及基因工程中TdT工具酶活力的测定.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.     

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT

Endogenous nuclease digestion of thymus nuclei from 3–4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70–85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15–30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.Abbreviations dNTP deoxyribonucleoside triphosphate - DTT dithiothreitol - Ig immunoglobulin - PMSF phenylmethylsulfonylfluoride - rNTP ribonucleoside triphosphate - TCR T cell receptor - TdT terminal deoxynucleotidyl transferase - VDJ variable, diversity and joining segments of Ig or TCR genes     

Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.

The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.     

An in situ study of B-lymphocytopoiesis in rat bone marrow. Topographical arrangement of terminal deoxynucleotidyl transferase-positive cells and pre-B cells

To understand bone marrow (BM) as a site of B-lymphocytopoiesis, insight into the topographical arrangement of developing B cells and their relationships to the microenvironment in vivo is required. To study the spatial distribution of B lymphocyte progenitors defined by intracellular markers (cytoplasmic mu H chain and nuclear terminal deoxynucleotidyl transferase (TdT], we developed a technique to cut frozen femurs of rat, yielding cross-sections with intact subendosteal and central marrow. By using (double) immunofluorescence staining techniques we located pre-B and TdT+ cells, and IgM+ B cells in those sections. Of the B cells present in BM, one-third was accumulated in the lumen of blood sinuses. The rest were in the BM parenchyma, as were virtually all pre-B and TdT+ cells. The subendosteal area was twice as rich in pre-B and TdT+ cells as the central area, and within the subendosteal area a profound positive gradient toward the bone was evident. B cells showed an equal distribution over the center and the periphery of the BM. The distribution patterns of B lineage cells in the BM parenchyma were analyzed and shown in part to deviate from random distribution. Additional study of clonal development and microenvironmental factors in hematopoiesis will have to clarify the underlying mechanisms for the observed distribution patterns of B cell precursors in BM.     

层析聚焦法分离纯化末端脱氧核苷酰转移酶及其抗血清制备和鉴定

 应用层析聚焦和Oligo(dT)-纤维素亲和层析相结合的方法,从小牛胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)。纯化的TdT聚丙烯酰胺凝胶电泳呈一条区带,SDS-聚丙烯酰胺凝胶电泳分子量为24,000及26,000d的两条区带。此纯化TdT径戊二醛交联法,自身交联后免疫家兔,得到兔抗小牛TdT的单价抗血清,并进行了免疫学鉴定。     

DNA polymerases of human tonsil and chicken bursa: absence of a distinct B cell specific terminal deoxynucleotidyl transferase.

The extracts obtained from chicken bursas and human tonsils, both of which are known to contain predominantly B lymphocyte precursor cells, were examined for the presence of putative B cell specific terminal deoxynucleotidyl transferase (B-TdT). Neither of the two types of tissue extracts revealed the presence of specific TdT, although the presence of DNA polymerase α, β, and γ could be easily demonstrated.     

猪胸腺末端脱氧核苷酰转移酶的分离纯化及性质的研究

 本文报道了一种较简便的从猪胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)的方法。经一次磷酸纤维素柱层析,使TdT与DNA聚合酶分离;经三次柱层析,可获得SDS-电泳纯,分子量约60K的产品。其酶学性质与牛胸腺TdT相似。