Bentolila L A, Fanton d'Andon M, Nguyen Q T, Martinez O, Rougeon F, Doyen N
Unité de Génétique et Biochimie du Développement, Paris Cédex 15, France.
EMBO J. 1995 Sep 1;14(17):4221-9. doi: 10.1002/j.1460-2075.1995.tb00096.x.
Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.
先前已在小鼠胸腺中鉴定出两种选择性剪接的末端脱氧核苷酸转移酶转录本，即TdTS和TdTL，它们分别编码509个和529个氨基酸的蛋白质。在此我们表明，骨髓来源的B淋巴细胞中也存在同样的这两种转录本。此外，我们证明在TdTL的羧基末端附近发现的相应20个氨基酸的插入显著改变了该酶的功能。与TdTS不同，TdTL不会在V(D)J位点特异性重组底物的重组连接处催化N区插入。为了解释N区插入缺失的原因，我们对区分TdT两种同工型的不同参数进行了表征。对转染细胞提取物的检测显示，TdTL向DNA 3'末端添加核苷酸的能力降低，这与较低的末端转移酶活性一致。此外，这些细胞中TdTL蛋白的半衰期比TdTS短2倍。最后，尽管TdTL与TdTS具有相同的核定位信号，但这两种同工型的细胞定位却显著不同。与核内的TdTS不同，TdTL仅存在于细胞质中。所有这些特性可能导致TdT两种同工型之间的功能差异。然而，TdTL自身的亚细胞定位就可以解释其无法添加N区的原因。
