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A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the X phage vector Xgtll. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had 3..galactosidase-cDNA fusion proteins identifiable after electrophoretic fractidhatioh by immunoblotting with anti-terminal transferase antibody. The iredosinant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyftae-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.Terminal deoxynucleotidyltransferase (TdT) is a unique DNA polymerase that without template direction catalyzes the addition of deoxyribonucleotides onto the 3'-hydroxyl ends of DNA primers (1-3). It is present in the immature fraction of thymocytes (4-7), in a small fraction of bone marrow cells (5, 8), in transformed pre-B-and pre-T-cell lines (9, 10), and in leukemic cells (6, 11). The enzyme purified by the method of Yoneda and Bollum (12) and Chang and Bollum (13) is a dimer of Mr 26,000 and Mr 8,000 chains. This structure is, however, an artifactual result of proteolytic cleavage during the purification. The enzyme is synthesized as a single chain of Mr 58,000 in mice and Mr 55,000-60,000 in humans (14-18).The function of TdT is not fully established. It was suggested that the enzyme might be responsible for somatic point mutation of immunoglobulin genes (19), but it now appears that somatic point mutation occurs late in B-lymphocyte maturation, when cells no longer contain TdT (20,21). A recent proposal that TdT might be responsible for inserting nucleotides (N regions) at VH-D and D-JH (22) junctions (junctions of heavy chain variable-diversity and diversityjoining region genes) has received experimental support (unpublished results). N-region insertion in a T-cell receptor chain may also occur (23) and is possibly a result of the relatively high TdT levels in thymocytes.We describe here the isolation from a thymoma cell line cDNA library of a clone that encodes TdT. Because the amino acid sequence of TdT has not been reported, there was no nucleic acid probe available for screening recombinants. Instead, specific antibodies raised against the purified twochain bovine enzyme, which cross-react with the murine enzyme, were used to probe a cDNA library constructed in the Xgtl1 expression system of Young and Davis (24). As well as providing a cDNA clone with which to characterize TdT, this work shows the feasibility of cloning ctlNA representations of rare mRNAs with conventional antibody reagents. METHODS Xgtl1 cDNA Library Construction. Double-stranded cDNA was synthesized from 20 gg of RL 11 poly(A)-...