Peterson R C, Cheung L C, Mattaliano R J, White S T, Chang L M, Bollum F J
J Biol Chem. 1985 Sep 5;260(19):10495-502.
A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.
一个与pT17相关的克隆DNA片段，包含人末端脱氧核苷酸转移酶的部分cDNA序列，被用作探针，在由人淋巴母细胞KM - 3 cDNA构建的λgt11文库中筛选该酶的全长cDNA序列。分离出一个含有2068个碱基对插入片段的重组体，并作为两个亚克隆pT711和pT106重新克隆到测序质粒pUC - 8的EcoRI位点。DNA测序和杂交研究表明，pT711包含pT17序列和另外172个上游核苷酸。pT711代表末端转移酶蛋白羧基端一半的编码序列。pT106含有一个965个碱基对的插入片段，在Northern印迹上与pT711杂交到相同的mRNA，并且包含一个与pT711中插入片段的阅读框同相位的开放阅读框。对小牛胸腺末端转移酶58 kDa肽段的氨基酸测序失败，表明N端被封闭。对该蛋白56 kDa形式的N端测序产生了24个氨基酸，对应于从pT106中插入片段的第398位残基开始翻译的人cDNA编码序列，牛和人序列之间的同源性为83%。起始密码子被指定为pT106中插入片段核苷酸329处的ATG序列。将翻译后的人末端转移酶序列与小牛胸腺酶的肽段进行比较，发现在所确定的263个氨基酸中，人和牛酶之间的同源性优于90%。pT106和pT711中的编码序列被重新克隆到表达质粒pUC - 19中，位于lac启动子下游，并与lac Z基因的编码序列同相位。携带人末端转移酶重建编码序列的细菌裂解物含有一种60 kDa的融合蛋白，该蛋白在免疫印迹上与兔抗末端转移酶抗体反应，并表现出酶活性。用抗人末端转移酶的小鼠单克隆抗体从细菌裂解物中分离这种融合蛋白，得到预期的60 kDa蛋白。
