Molecular cloning of human terminal deoxynucleotidyltransferase.

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Abstract
A c[complementary]DNA of the human terminal deoxynucleotidyltransferase (TdT; terminal transferase, ED 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in .lambda.gt11 by using immunological procedures. Four inserts containing 723-939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to 58-kDa [kilodalton] 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 [high performance liquid chromatography] 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 3 regions beginning after Arg that have > 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence was cloned.