Peterson R C, Cheung L C, Mattaliano R J, Chang L M, Bollum F J
Proc Natl Acad Sci U S A. 1984 Jul;81(14):4363-7. doi: 10.1073/pnas.81.14.4363.
A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned.
通过免疫学方法，从λgt 11载体中的人淋巴母细胞cDNA文库中分离出人类末端脱氧核苷酸转移酶（TdT；“末端转移酶”，EC 2.7.7.31）的cDNA。将4个含有723至939个碱基对的插入片段亚克隆到pBR322中，用于杂交和初步序列研究。通过与重组DNA杂交选择的mRNA被翻译成一种58 kDa的肽，该肽能与抗小牛末端转移酶的兔抗体和抗人末端转移酶的小鼠单克隆抗体发生特异性免疫沉淀。用切口平移的pBR322重组DNA对KM3（TdT+）细胞的总poly（A）+ RNA进行印迹杂交，检测到一条约2000个核苷酸的信使RNA，足以编码该蛋白质中的580个氨基酸。来自末端转移酶阴性细胞的mRNA在杂交选择或RNA印迹杂交中均未给出信号。从克隆于pUC8的缺失片段中获得了939个碱基对插入片段序列的完整序列。该DNA序列包含一个编码238个氨基酸的开放阅读框，约占该蛋白质的40%。通过HPLC从琥珀酰化的58 kDa小牛胸腺末端转移酶的胰蛋白酶消化物中分离出的三个肽段进行了测序，提供了20、18和22个氨基酸残基的肽段序列。对939个碱基对插入片段的翻译序列进行搜索，发现从精氨酸之后开始的三个区域与从小牛胸腺末端转移酶肽段确定的序列具有大于90%的同源性。这些结果提供了明确的证据，表明已克隆出人类末端转移酶序列。
