ISOLATION OF P1-BACTERIOPHAGE CLONES CONTAINING LARGE CONTIGUOUS SEGMENTS OF THE HUMAN AND MOUSE LOCI FOR THE T-CELL CORECEPTOR MOLECULE-CD8

The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and C D8 beta chains) and CD4 undergo a complex pattern of regulated express ion during T-cell maturation. In the thymus, the most immature cells p rogress from expressing neither molecule (the dobule-negative [DN] sta ge) to an intermediate stage at which both are coexpressed (the double -positive [DP] stage). As a result of thymic selection and further dif ferentiation, DP cells give rise to the most mature thymic cells and p eripheral T cells that express either CD8 or CD4 (the single-positive [SP] stage). Our previous studies of the transcriptional regulatory me chanisms controlling CD8 alpha expression during the DN --> DP and DP --> SP transitions suggest the existence of important cis-acting eleme nts located a considerable distance from the CD8 alpha gene and that t hese elements might serve to regulate both CD8 alpha and CD8 beta. Whi le both genes and intergenic DNA span similar to 60 kb in the mouse, t he relevant cis elements could lie either within or beyond this region . As a result, we sought to isolate large contiguous segments of DNA i n P1 bacteriophage that covered at least this region from the mouse an d human CD8 locus. Our initial physical characterization of these clon es demonstrates the value of the PI system as all isolated clones were found to contain single contiguous 85- to 95-kb segments of DNA that are faithful replicas of the chromosomal locus. The presence of abunda nt native flanking DNA both upstream and downstream of the intact codi ng regions will make these clones extremely useful for identifying phy siological CD8 cis-active regulatory elements by virtue of their abili ty to direct appropriate lineage- and stage-specific expression in tra nsfected and transgenic T cells.