Cloning and comparative analysis of the human pre-T-cell receptor a-chain gene

Abstract : In immature T cells the T-cell receptor (TCR) 18-chain gene is rearranged and expressed before the TCR a-chain gene. At this stage TCR 18 chain can form disulfide-linked heterodimers with the pre-T-cell receptor a chain (pTa). Using the recently isolated murine pTa cDNA as a probe, we have isolated the human pTa cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTa with the mouse pTa molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTa gene is expressed in immature but not mature T cells and is located at the p2l.2-pl2 region of the short arm of chromosome 6. Intrathymic T-cell development is accompanied by ordered rearrangement of T-cell receptor (TCR) genes as well as by changes in expression of surface markers (1). At a certain developmental stage, CD4-, CD8-, CD3l0w, CD44+, CD25+ T-cell precursors downregulate CD44 expression and begin to rearrange the TCR (3-chain (TCRI3) locus. After productive TCR(3 genes are formed, thymocytes become CD25-, acquire CD4/CD8 coreceptors on the cell surface, and rearrange the TCRa locus (2-5). At the CD4+ CD8+ TCRaf+ stage, thymocytes are subjected to positive and negative selection depending on the quality of TCR-major histocompatibility complex ligand interaction (6, 7). While the TCRao3 controls late thymic development, early developmental steps are controlled by the pre-TCR composed of the variant TCR,3 chain that is disulfide linked to the invariant pre-TCRa (pTa) protein recently described in mice (7-9). The TCRf3-pTa heterodimer is associated with CD3 molecules (10) and signals triggered by the pre-TCR induce expansion and differentiation of immature precursor cells (8, 9, 11). Here we report on the isolation and characterization of the human pTa cDNA and its comparison with the murine homologue.1 In addition we provide data on the chromosomal location as well as on pTa expression in thymic subsets. MATERIALS AND METHODS cDNA Isolation and Sequencing. A 3-year-old human thy-mus cDNA library constructed in AgtlO vector (1 x 106 clones) (Clontech; HL1 127A) was screened with a probe corresponding to the full-length murine pTa. Prehybridization and hy-bridization were done at 65°C in a solution containing 1 M NaCl, 50 mM Tris HCl (pH 7.2), 10% dextran sulfate, 1% SDS, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and 0.250 mg of salmon sperm DNA per ml. Filters were washed in 2x SSC first at room temperature and then at 65°C. The murine cDNA was used as a probe and labeled with
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Paola Porto, Ludovica Brunot, Marie-Genevieve Mattei, Harald von Boehmer, Claude Saint-Ruf. Cloning and comparative analysis of the human pre-T-cell receptor a-chain gene. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 1995, 92, pp.12105 - 12109. ⟨10.1073/pnas.92.26.12105⟩. ⟨inserm-02177298⟩

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