Zhang X L, Lee K L, Heng H H, Tsui L C, Parnes J R, Shepherd N S, Chamberlain J W
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Genet Anal Tech Appl. 1994;11(5-6):129-39. doi: 10.1016/1050-3862(94)90033-7.
The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and CD8 beta 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 CD8 alpha expression during the DN-->DP and DP-->SP transitions suggest the existence of important cis-acting elements located a considerable distance from the CD8 alpha gene and that these elements might serve to regulate both CD8 alpha and CD8 beta. While both genes and intergenic DNA span approximately 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.
T淋巴细胞共受体分子CD8（由CD8α和CD8β链组成）和CD4在T细胞成熟过程中经历复杂的调控表达模式。在胸腺中，最不成熟的细胞从不表达这两种分子（双阴性[DN]阶段）发展到同时表达这两种分子的中间阶段（双阳性[DP]阶段）。由于胸腺选择和进一步分化，DP细胞产生最成熟的胸腺细胞和外周T细胞，这些细胞表达CD8或CD4（单阳性[SP]阶段）。我们之前对DN→DP和DP→SP转变过程中控制CD8α表达的转录调控机制的研究表明，存在距离CD8α基因相当远的重要顺式作用元件，并且这些元件可能用于调控CD8α和CD8β。虽然在小鼠中这两个基因和基因间DNA跨度约为60kb，但相关的顺式元件可能位于该区域之内或之外。因此，我们试图在P1噬菌体中分离出至少覆盖小鼠和人类CD8基因座这一区域的大片连续DNA片段。我们对这些克隆的初步物理特征分析证明了P1系统的价值，因为所有分离的克隆都被发现包含单一连续的85至95kb DNA片段，这些片段是染色体基因座的忠实复制品。完整编码区上下游存在丰富的天然侧翼DNA，这将使这些克隆对于通过其在转染和转基因T细胞中指导适当的谱系和阶段特异性表达的能力来鉴定生理性CD8顺式活性调控元件极为有用。
