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。     

Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.     

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。     

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.     

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.     

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.     

High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Terminal deoxynucleotidyltransferase containing megadalton complex from young rat thymus nuclei: identification and characterization

Nuclear matrix prepared from 2-3 week old rat thymuses contains tightly bound TdT activity which has been quantitatively solubilized with nonionic detergent and sonication. TdT is contained in a discrete complex with a sedimentation value of 23 S. The complex is retained on an anti-TdT antibody column and contains DNA ligase and 3'-5' exonuclease activities as well as DNA and several other proteins but is devoid of replicative DNA polymerases. Such a type of multienzyme complex is absent from the nuclear extracts of thymus prepared from older rats and also from liver and spleen extracts of young and old rats.     

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.     

Non-germ-line elements (NGE) are present in the T cell receptor beta-chain genes isolated from the mutant mouse, motheaten (me/me)

Extra nucleotides (which we call NGE, for non-germ-line elements) are inserted at the junctions of rearranged V, D, and J segments in the immunoglobulin heavy and T cell receptor beta-chain V region genes. NGE addition helps diversity HV3 regions of these genes. It is believed that NGE are added enzymatically and without template during the joining process. Terminal deoxynucleotidyl transferase (TdT) is thought to be the cause of NGE formation. TdT is normally detected in murine thymus and bone marrow cells, but its presence in the immunodeficient mutant mouse, Motheaten (me/me), is extremely reduced in these tissues. To determine whether this TdT deficiency could affect NGE formation during the V-D-J joining of antigen receptor genes, we cloned several rearranged T cell receptor beta-chain genes from thymocytes of me/me mice. Our sequence analysis revealed that Motheaten thymus beta-chain genes have approximately 4 base pair NGE, which are comparable in size to the NGE of wild-type genes. These results do not support the idea that TdT is the NGE-forming enzyme, although it is still possible that a low but residual level of TdT is capable of NGE formation in Motheaten. Alternatively, our data may suggest that the TdT activity in Motheaten T cells is normal; however, the number of TdT+ cells is greatly reduced in the Motheaten thymus, due to a severe defect in T cell development.     

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3–4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70–85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15–30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20–25% of the nuclear DNA polymerase was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replicationvia-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.Abbreviations dNTP deoxyribonucleoside triphosphate - DTT dithiothreitol - Ig immunoglobulin - PMSF phenylmethylsulfonylfluoride - rNTP ribonucleoside triphosphate - SDS sodium dodecyl sulphate - TCR T cell receptor - TdT terminal deoxynucleotidyl transferase - VDJ variable, diversity and joining segments of Ig or TCR genes     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage

Anti-CD3 (T3) Ab reacting with different proportions of thymocytes (anti-CD3a: UCHT1, anti-CD3b: T10B9, and anti-CD3c: OKT3) were tested for cytoplasmic (cCD3) and membrane (mCD3) expression in the bone marrow, thymus, and blood in man and selected primates. The expression of cCD3a and cCD3c in the perinuclear and Golgi area of large, BrdU-incorporating, strongly TdT+ thymic blasts probably represents one of the earliest signs of T cell commitment, because these blast cells are CD1-, CD4-, CD8-, and mCD3-. The cCD3+, TdT+ cells are normally restricted to the thymus and are absent among the TdT+ cells of bone marrow. The anti-CD3b Ab used, T10B9, co-caps and co-modulates with the other anti-CD3 Ab and is a T cell-specific reagent at a membrane level but does not bind to perinuclear cCD3. Instead, this reagent cross-reacts with a filamentous cytoplasmic network in non-T cells in man and in primates S. oedipus and M. rhesus despite their T cell negativity for mCD3. The characteristics of all T-ALL cases studied: cCD3+, CD7+ along with nuclear TdT+ suggest lineage fidelity to early thymic blasts. As a marked contrast, cCD3 is absent in common ALL and in AML, including cases that concomitantly express CD7 and myeloid antigens. Thus, the cCD3, TdT combination provides a very sensitive assay for residual T-ALL blasts outside the normal thymus.     

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     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.     

Identification of subsets of proliferating low Thy 1 cells in thymus cortex and medulla

Subsets of proliferating thymocytes were identified in the normal mouse thymus by in vivo labeling with [³H]TdR and by cell separation according to relative amounts of Thy 1 antigen. In order to resolve apparent discrepancies in the literature, parenteral and topical application of [³H]TdR were compared as labeling methods for dividing thymocytes, and limited complement lysis and fluorescence-activated cell sorting were compared as separation principles for high Thy 1 and low Thy 1 thymocyte subsets. The separated cells were further characterized by immunofluorescence for terminal deoxynucleotidyltransferase (TdT), which normally is restricted to cortical thymocytes, and for H2 alloantigens, which are preponderant on medullary thymocytes. Four subsets of proliferating cortical thymocytes were identified after application of [³H]TdR to the thymus capsule. The major subset, which comprised about 92% of dividing cortical thymocytes, had a high Thy 1, low H2 phenotype. Most were also TdT + ve. The three minor subsets of proliferating cortical thymocytes each had a low Thy 1 phenotype, but differed according to H2 and TdT markers. Systemic injection of [³H]TdR also labeled the above subsets of dividing cortical thymocytes, but in addition it detected a subset of proliferating low Thy 1, low H2, TdT — ve cells in the thymus medulla. The latter subset comprised about one-third of the pool of proliferating low Thy 1 cells. In their aggregate the four subsets of low Thy 1 cells constituted approximately 13% of total proliferating thymocytes and 1.1% of total thymocytes. The identification of discrete subsets of proliferating low Thy 1 cells in the thymus cortex as well as in the thymus medulla is compatible with the hypotheses that all thymocytes are descended from low Thy 1 precursors and that separate precursor cell subsets exist for cortical and medullary thymocytes.     

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The K_m for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg²⁺ or Mn²⁺, whereas the K_m for the initiator, poly(deoxyadenylic acid $\text{[poly(d(pA)}\text{50}\text{)]}$, with an average chain length of 50 residues, is 2.5 μm in the presence of Mg²⁺ and 0.3 μm in the presence of Mn²⁺. The maximum velocity is higher for the calf thymus TdT in the presence of Mg²⁺ than in Mn²⁺. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn²⁺ than with Mg²⁺. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.