Use of D gene segments with irregular spacers in terminal deoxynucleotidyltransferase (TdT)+/+ and TdT-/- mice carrying a human Ig heavy chain transgenic minilocus.

Abstract

D gene segments with irregular spacers (DIR) are D gene segments that are specific to higher primates. Their use is controversial because of their G+C-rich long sequences. In the human, it has always been tempting to assume that a complementarity-determining region 3 sequence has been added by terminal deoxynucleotidyltransferase (TdT) activity and is not derived from DIR recombination. Herein, we examine the use of human DIR gene segments by cross-breeding the human Ig heavy chain minilocus pHC1 transgenic mice and TdT-deficient mice. In the absence of TdT and with a defined set of human D gene segments, it is relatively easy to demonstrate that DIR2 is used to form human Ig heavy chains, contributing to 7% of the human heavy chain rearrangements. VHDJH rearrangements (where H is heavy chain) in the minilocus TdT-/- mice use small portions of DIR2 located throughout the coding sequence. These results constitute the strongest evidence to date that DIR gene segments are used to form human antibodies. Additionally, we show that direct and inverted DIR2JH and VHDIR2 rearrangements occur in the minilocus transgenic mice. During these rearrangements, DM2 3' signal sequence and a new DIR2 5' signal sequence are used. These rearrangements generally follow the 12/23 recombination rule. Our results at the VHDJH, DJH, and VHD levels indicate that DIR2 is used to form human heavy chains in transgenic mice. The rearrangement of this gene segment likely involves, however, other mechanisms in addition to the classical VHDJH recombination.