Isolation of P1 bacteriophage clones containing large contiguous segments of the human and mouse loci for the T-cell coreceptor molecule CD8

Abstract

The T-lymphocyte coreceptor molecules CD8 (composed of CD8α and CD8β chains) and CD4 undergo a complex pattern of regulated expression during T- cell maturation. In the thymus, the most immature cells progress from expressing neither molecule (the double-negative [DN] stage) to an intermediate stage at which both are coexpressed (the double-positive [DP] stage). As a result of thymic selection and further differentiation, DP cells give rise to the most mature thymic cells and peripheral T cells that express either CD8 or CD4 (the single-positive [SP] stage). Our previous studies of the transcriptional regulatory mechanisms controlling CD8α expression during the DN → DP and DP → SP transitions suggest the existence of important cis- acting elements located a considerable distance from the CD8α gene and that these elements might serve to regulate both CD8α and CD8β. While both genes and intergenic DNA span ~60 kb in the mouse, the relevant cis elements could lie either within or beyond this region. As a result, we sought to isolate large contiguous segments of DNA in P1 bacteriophage that covered at least this region from the mouse and human CD8 locus. Our initial physical characterization of these clones demonstrates the value of the P1 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 abundant native flanking DNA both upstream and downstream of the intact coding regions will make these clones extremely useful for identifying physiological CD8 cis-active regulatory elements by virtue of their ability to direct appropriate lineage- and stage-specific expression in transfected and transgenic T cells.