Molecular cloning of human terminal deoxynucleotidyltransferase.

Abstract

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.