Cecilie Boysen, Inyoul Lee, Kai Wang, and Leroy Hood.
Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195.
The T cell receptors are heterodimers containing two subunits, alpha/beta or gamma/delta, recognizing specific antigenic peptides embedded in molecules encoded by the major histocompatibility complex on antigen-presenting cells. Upon recognition, the short cytosolic tails of the two antigen binding subunits transmit the signal via other T cell receptor components to the interior of the T cell, where appropriate responses occur. The T cell receptor antigen-binding chains are encoded by three different loci: alpha/delta, beta, and gamma. These loci have a very special organization of their genes in order to be able to generate large repertoires of different T cell receptors.
We have sequenced 920 kb of the alph/delta locus, and anticipate to finish the last two gaps (~100 + 20 kb) later this year. Initially, we sequenced overlapping cosmids derived from YACs, but later mapped the region with BACs, which we sequenced directly applying shotgun sequencing techniques. To date, we have sequenced 5 BACs, ranging in size from 80 kb to 210 kb. These constitute the bulk of the total sequence. Phil Green in our department has developed an assembling program, Phrap, which enabled us to assemble the shotgun BAC inserts without significant problems. BACs are also an attractive alternative to YACs for mapping. BAC clones are much easier to handle than YAC clones; they are rarely chimeric, and show little, if any, rearrangement. For these reasons, we have now chosen to sequence the mouse T cell receptor alpha/delta region using BACs.
The sequence obtained for the human T cell receptor alpha/delta locus has provided insights into the structure, organization, and evolution of these T cell receptor elements. It allows generation of STSs across any region, as is useful when looking for polymorphisms in connection with either genes or microsatellites. Furthermore, interesting distributions of genome-wide repeats, such as Alu or LINE sequences, have been observed over this region. Initial comparison with a ~130 kb region sequenced in mouse reveals striking sequence similarity, not only for the coding features, but also for the intergenic sequence.