ENHANCER ELEMENTS IN B-LYMPHOCYTES AND T-LYMPHOCYTES

这个项目最近的一个焦点是3‘Long Open的作用 人T细胞白血病病毒I型的读框 它编码一个40kD的蛋白(P40x)。这种蛋白质是阳性的 调控HTLV-I长末端的转录 重复(LTR)在一种被称为反式激活的现象中。我们 已经成功表达了完整的p40x编码序列 在大肠杆菌和杆状病毒载体中。这两种p40x蛋白都是 能够刺激HTLV-I LTR的转录。 P40x蛋白已经得到了显著的纯化。 我们无法确定任何特定于序列的DNA 与p40x结合的特性，表明该蛋白被激活 HTLV-1启动子间接使用细胞 转录因子。我们的目标是了解 P40x蛋白反式激活的生化机制 而细胞转录因子参与了这一过程 进程。不同的转录调控序列位于 已确定HTLV-I LTR的上游序列 并通过化学方法合成。这些序列具有 增强子序列的性质，已经被克隆并被反式- 被HTLV-I p40x蛋白激活。使用一种新的DNA-蛋白质 在这个实验室开发的交联协议，我们有 确定了与21个碱基p40x相互作用的细胞因子- 反应顺序。我们已经证明，通过突变 分析表明，细胞蛋白在体外的结合 与p40x在体内的生物学活性有关 反式激活。

A recent focus of this project has been the role of the 3' long open reading frame of the human T-cell leukemia virus type-I (HTLV-I) which encodes a 40-Kd protein (p40x). This protein positively regulates transcription directed by the HTLV-I long terminal repeat (LTR) in a phenomenon known as trans-activation. We have succeeded in expressing the complete p40x coding sequence in E. coli and in a baculovirus vector. Both p40x proteins are capable of stimulating transcription from the HTLV-I LTR. Significant purification of the p40x proteins has been achieved. We have been unable to attribute any sequence-specific DNA binding properties to p40x, suggesting that the protein activates the HTLV-1 promoter in an indirect fashion using cellular transcription factors. Our objective is to understand the biochemical mechanism of trans-activation by the p40x protein and the involvement of cellular transcription factors in this process. Distinct transcriptional regulatory sequences located in the upstream sequences of the HTLV-I LTR have been identified and chemically synthesized. These sequences, which have the properties of enhancer sequences, have been cloned and are trans- activated by the HTLV-I p40x protein. Using a novel DNA-protein cross-linking protocol developed in this laboratory, we have identified cellular factors that interact with the 21 bp p40x- responsive sequence. We have demonstrated, by mutational analysis, that the binding of the cellular proteins in vitro correlates with the in vivo biological activity in response to p40x trans-activation.