ENHANCER ELEMENTS IN B-LYMPHOCYTES AND T-LYMPHOCYTES

这个项目最近的一个重点是3'长开的作用 人类T细胞白血病病毒I型（HTLV-I）的阅读框架 其编码40-Kd蛋白（p40 x）。 这种蛋白质 调节HTLV-I长末端指导的转录 重复序列（LTR）在一种称为反式激活的现象。 我们 成功表达了完整的p40 x编码序列 在大肠coli和杆状病毒载体中。 两种p40 x蛋白都是 能够刺激HTLV-1 LTR的转录。 已经实现了p40 x蛋白的显著纯化。 我们无法将任何序列特异性DNA 与p40 x的结合特性，表明该蛋白质激活 HTLV-1启动子以间接的方式使用细胞 转录因子 我们的目标是了解 p40 x蛋白反式激活的生化机制 以及细胞转录因子在其中的参与 过程 不同的转录调控序列位于 已经鉴定了HTLV-1 LTR的上游序列 和化学合成。 这些序列， 增强子序列的性质，已被克隆，并被反式- 由HTLV-I p40 x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 鉴定了与21 bp p40 x相互作用的细胞因子， 响应序列 我们已经证明，通过突变 分析，细胞蛋白在体外的结合 与体内对p40 x应答的生物活性相关 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.