In condizioni fisiologiche la TdT è espressa esclusivamente da precursori linfoidi B e T nel timo (e nel midollo osseo), con regolazione genica mediata da un promotore tessuto-specifico.

Nello schema differenziativo dei linfociti B e T  l'enzima TdT esercita la sua attività di DNA-polimerasi precocemente, durante i fenomeni di riarrangiamento dei geni che codificano per il T-cell receptor ed i geni delle immunoglobuline. L'attività della TdT, che agisce come DNA-polimerasi aggiungendo nucleotidi indipendentemente dalla presenza di un DNA template, contribuisce alla diversità dei recettori antigenici per i linfociti B e T, determinando la formazione delle regioni non-germline (N-regions). Comunque la funzione della TdT, dal punto immuno-fisiologico, non costituisce un difetto fondamentale, come dimostrato nei topi privi di TdT .

 

Regulation of terminal deoxynucleotidyl transferase gene expression in mice and men.
Coleman MS, Yang B, Sorscher D.

Crit Rev Eukaryot Gene Expr 1992;2(3):237-50

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.

A nontemplate directed DNA polymerase, terminal deoxynucleotidyl transferase (terminal transferase) is expressed in a tissue-specific and development stage-specific manner. Its enzymatic properties and tissue localization have implicated the protein in development of normal immune function. Significant progress has been made in understanding the enzymology and important domains of this protein. More recently, studies have focused on regulation of the gene that codes for the protein in mice and humans. The murine gene has yielded to these studies more readily than the human gene. A murine basal promoter element has been identified along with several trans-acting protein factors that may regulate gene expression. In the human gene there is no evidence for a basal promoter element. Rather, the promoter exhibits tissue specific properties. The present article reviews recent developments in this field.

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

Yang-Feng TL, Landau NR, Baltimore D, Francke U.
Cytogenet Cell Genet 1986;43(3-4):121-6

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.

 

T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis.
Bonati A, Zanelli P, Ferrari S, Plebani A, Starcich B, Savi M, Neri TM.
Blood 1992 Mar 15;79(6):1472-83

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta-chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D-J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti-calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta-chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.

 

Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.
Desiderio SV, Yancopoulos GD, Paskind M, Thomas E, Boss MA, Landau N, Alt FW, Baltimore D.
Nature 1984 Oct 25-31;311(5988):752-5

The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.

Mice lacking TdT: mature animals with an immature lymphocyte repertoire.
Gilfillan S, Dierich A, Lemeur M, Benoist C, Mathis D.
Science 1993 Aug 27;261(5125):1175-8

In adult animals, template-independent (or N) nucleotides are frequently added during the rearrangement of variable (V), diversity (D), and joining (J) segments of lymphocyte receptor genes, greatly enhancing junctional diversity. Receptor genes from adult mice carrying a mutation in the terminal deoxynucleotidyl transferase (TdT) gene have few N nucleotides, providing proof that this enzyme is essential for creating diversity. Unlike those from normal adults, receptor genes from adult mutant mice show extensive evidence of homology-directed recombination, suggesting that TdT blocks this process. Thus, switch-on of the TdT gene during the first week after birth provokes an even greater expansion of lymphocyte receptor diversity than had previously been thought.

 

Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes.
Komori T, Okada A, Stewart V, Alt FW.
Science 1993 Aug 27;261(5125):1171-5

During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.

  Scheda a cura di Marco Chilosi (GYM)