Yang B, Gathy K N, Coleman M S
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill 27599-7260, USA.
Nucleic Acids Res. 1995 Jun 11;23(11):2041-8. doi: 10.1093/nar/23.11.2041.
A cDNA clone coding for avian terminal deoxynucleotidyl transferase (TdT) has been isolated and sequenced. The size of this cDNA was 2545 bp with an open reading frame of 1521 bp and a predicted translation product of 58 kDa. Comparison of this TdT sequence with other known TdT sequences has revealed a very high degree of homology at both the DNA and predicted amino acid levels. The chicken TdT cDNA was expressed in a bacterial system and the protein was purified by affinity chromatography. The purified recombinant enzyme, with a specific activity of approximately 1700 U/mg protein, was significantly less active than TdTs from mammalian species. This finding correlates with the observation that TdT isolated from avian thymus has lower activity than that isolated from any mammalian thymus source. Northern blot hybridization analyses and reverse transcription PCR of RNA preparations were carried out with the chicken cDNA. The data generated from these experiments revealed that the TdT RNA was only expressed in the thymus and not in the bone marrow or the bursa of Fabricius during pre- and post hatching chicken development. These data suggest that while TdT is probably involved in N region addition in chicken T-cell receptor genes, it is unlikely to play a role in diversification of immunoglobulin genes.
编码禽末端脱氧核苷酸转移酶（TdT）的一个cDNA克隆已被分离并测序。该cDNA大小为2545 bp，有一个1521 bp的开放阅读框，预测的翻译产物为58 kDa。将此TdT序列与其他已知的TdT序列进行比较，发现在DNA和预测的氨基酸水平上均有很高的同源性。鸡TdT cDNA在细菌系统中表达，蛋白质通过亲和层析纯化。纯化的重组酶比活约为1700 U/mg蛋白质，其活性明显低于来自哺乳动物的TdT。这一发现与从禽胸腺分离的TdT活性低于从任何哺乳动物胸腺来源分离的TdT这一观察结果相关。用鸡cDNA对RNA制剂进行了Northern印迹杂交分析和逆转录PCR。这些实验产生的数据表明，在孵化前和孵化后的鸡发育过程中，TdT RNA仅在胸腺中表达，而不在骨髓或法氏囊中表达。这些数据表明，虽然TdT可能参与鸡T细胞受体基因的N区添加，但它不太可能在免疫球蛋白基因的多样化中发挥作用。
