VP16 AND HOMEODOMAIN-MEDIATED TRANSCRIPTIONAL REGULATION

这个提案的重点是蛋白质--蛋白质和蛋白质--DNA 病毒转录因子VP16与单纯疱疹病毒的相互作用 病毒病毒体成分，在感染后，负责增强 病毒即刻早期基因的表达。 这个的目标 建议有两个方面。 首先，他们是针对理解， 详细描述了蛋白质-蛋白质和蛋白质-DNA相互作用，其中VP 16 参与，涉及人同源结构域蛋白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，它们被设计来确定类似的机制是否 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， VP16可以识别Oct-1同源结构域，并且VP16和Oct-1结合 TAATGARAT序列内的相邻顺式子元件。 研究 具体目标1中所述的方法使用足迹法和蛋白质-DNA 交联技术来鉴定Oct-1识别的序列， TAATGARAT顺式元件内的VP16。 具体描述的研究 目的2旨在了解VP16功能的结构基础。 在其他技术中，他们将采用定点诱变， 确定负责与Oct-1相关的VP16区域 同源结构域、HCF和DNA。并验证一个假设， VP16亚结构域负责Oct-1同源结构域识别。 具体目标3涉及目前已知的HCF的身份 仅作为色谱级分。 HCF将从哺乳动物中纯化 和果蝇提取物通过蛋白质亲和性和 常规色谱技术。 或者，如果纯化 证明困难，编码HCF活性的cDNA将通过遗传学方法克隆。 酵母中的互补作用 最后，具体目标4旨在测试 假设类似于VP16和Oct-1的相互作用 同源结构域在正常细胞转录期间发生。 这些研究 将基于10月1日参与的VP16区域的鉴定 同源结构域关联;如果细胞转录因子发挥功能， 类似于VPI6的存在，这种同源结构域识别亚结构域可以 在他们中间被保存了下来。 将采用两种策略， 利用这一潜在的保护。 首先，PCR技术将 用于鉴定细胞cDNA中相似的蛋白质编码区，和 第二，针对VP16同源结构域识别产生的抗血清 亚结构域将用于筛选cDNA表达文库。 如果这两 策略证明是不成功的，另一种方法使用一种新的 融合噬菌体cDNA克隆技术将用于蛋白质的检测 可以与同源结构域相互作用。

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.