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Nucleic Acids Research, 1995, Vol. 23, No. 11 2041-2048
© 1995

MOLECULAR BIOLOGY

T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme

Baoli Yang*, Karen N. Gathy and Mary Sue Coleman+

Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7260, USA

* To whom correspondence should be addressed at present address: Department of Pathology, CB& 7525, 703 Brinkhouse-Bullitt Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA

Received January 6, 1995. Revised April 22, 1995. Accepted April 22, 1995.

A cDNA clone coding for avian terminal deoxy-nucleotidyl transferase (TdT) has been isolated and sequenced. The size of this cDNA was 2545 bp with an open reading frame of 1521 bp and a predicted translation product of 58 kDa. Comparison of this TdT sequence with other known TdT sequences has revealed a very high degree of homology at both the DNA and predicted amino acid levels. The chicken TdT cDNA was expressed in a bacterial system and the protein was purified by affinity chromatography. The purified recombinant enzyme, with a specific activity of ~1700 U/mg protein, was significantly less active than TdTs from mammalian species. This finding correlates with the observation that TdT isolated from avian thymus has lower activity than that isolated from any mammalian thymus source. Northern blot hybridization analyses and reverse transcription PCR of RNA preparations were carried out with the chicken cDNA. The data generated from these experiments revealed that the TdT RNA was only expressed in the thymus and not in the bone marrow or the bursa of Fabricius during pre- and post hatching chicken development. These data suggest that while TdT is probably Involved In N region addition in chicken T-cell receptor genes, it is unlikely to play a role in diversification of immunoglobulin genes.

+Present address: Office of The Provost and Vice President for Academic Affairs, Scholes Hall 235, The University of New Mexico, Albuquerque, NM 87I3I-1OO2, USA


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