VP16 AND HOMEODOMAIN-MEDIATED TRANSCRIPTIONAL REGULATION

该提案重点关注蛋白质-蛋白质和蛋白质-DNA 病毒转录因子 VP16（一种单纯疱疹病毒）的相互作用 病毒病毒颗粒成分，在感染后负责增强 病毒立即早期基因的表达。 本次活动的目标 提案有两个方面。 首先，它们旨在理解 详细介绍了蛋白质-蛋白质和蛋白质-DNA 相互作用，其中 VP 16 参与，涉及人类同源域蛋白 Oct-1 的相互作用， 另一种称为 HCF 的宿主细胞蛋白和 TAATGARAT 顺式元件。 其次，它们旨在确定是否有类似的机制 在正常的细胞转录过程中起作用。 后面的实验 将以最近的研究结果为指导，这些研究表明 VPI6可以识别Oct-1同源域，并且VP16和Oct-1结合 TAATGARAT 序列中相邻的顺式子元件。 研究 具体目标 1 中描述的采用足迹法和蛋白质 - DNA 交联技术来识别 Oct-1 识别的序列和 TAATGARAT 顺式元件内的 VP16。 具体描述的研究 目标 2 旨在了解 VP16 功能的结构基础。 除其他技术外，他们将采用定点诱变来 确定 VP16 负责与 Oct-1 关联的区域 同源结构域、HCF 和 DNA。并检验一个独立的假设 VP16 子域负责 Oct-1 同源域识别。 具体目标 3 涉及目前已知的 HCF 身份 仅作为色谱分数。 HCF将从哺乳动物中纯化 和果蝇提取物通过蛋白质亲和力和 常规色谱技术。 或者，如果纯化 事实证明是困难的，编码HCF活性的cDNA将通过遗传克隆 酵母中的互补作用。 最后，特定目标 4 旨在测试 假设相互作用类似于 VP16 和 Oct-1 同源域发生在正常细胞转录过程中。 这些研究 将根据Oct-1涉及的VP16区域的识别 同源域协会；细胞转录因子是否发挥作用 类似于 VPI6 的存在，这个同源域识别子域可以 其中都得到了保存。 将采用两种策略 利用这种潜在的保护。 首先，PCR技术将 用于识别细胞 cDNA 中相似的蛋白质编码区，以及 第二，针对 VP16 同源域识别而产生的抗血清 子域将用于筛选 cDNA 表达文库。 如果这两个 策略被证明不成功，使用新颖的替代方法 “融合噬菌体”cDNA克隆技术将用于检测蛋白质 可以与同源域相互作用。

This proposal focuses on the protein--protein and protein--DNA interactions of a viral transcription factor, VP16, a herpes simplex virus virion component that, upon infection, is responsible for boosting expression of the viral immediate-early genes. The goals of this proposal are two-fold. First, they are directed at understanding in detail the protein--protein and protein--DNA interactions in which VP 16 participates, Interactions involving the human homeodomain protein Oct-1, another host cell protein termed HCF, and TAATGARAT cis-elements. Second, they are designed to determine whether analogous mechanisms operate during normal cellular transcription. The latter experiments will be guided by the results of recent studies which have shown that VPI6 can recognize the Oct-1 homeodomain, and that VP16 and Oct-1 bind adjacent cis-subelements within TAATGARAT sequences. The studies described in Specific Aim 1 employ footprinting and protein--DNA crosslinking techniques to identify the sequences recognized by Oct-1 and VP16 within TAATGARAT cis-elements. The studies described in Specific Aim 2 are aimed at understanding the structural basis of VP16 function. Among other techniques, they will employ site-directed mutagenesis to identify the regions of VP16 responsible for association with the Oct-1 homeodomain, HCF, and DNA. and test the hypothesis that an independent VP16 subdomain is responsible for Oct-1 homeodomain recognition. Specific Aim 3 is concerned with the identity of HCF, presently known only as chromatographic fraction. HCF will be purified from mammalian and Drosophila extracts by a combination of protein-affinity and conventional chromatographic techniques. Alternatively, if purification proves difficult, cDNA's encoding HCF activity will be cloned by genetic complementation in yeast. Lastly, Specific Aim 4 is designed to test the hypothesis that interactions analogous to those of VP16 and the Oct- 1 homeodomain occur during normal cellular transcription. These studies will be based on the identification of the VP16 region involved in Oct-1 homeodomain association; if cellular transcription factors that function analogously to VPI6 exist, this homeodomain-recognition subdomain may have been conserved among them. Two strategies will be employed to exploit this potential conservation. First, PCR techniques will be utilized to identify similar protein-coding regions in cellular cDNA, and second, antisera raised against the VP16 homeodomain-recognition subdomain will be used to screen cDNA expression libraries. If these two strategies prove unsuccessful, an alternative approach using a novel "fusion-phage" cDNA cloning technique will be employed to detect proteins that can interact with homeodomains.