VP16 AND HOMEODOMAIN-MEDIATED TRANSCRIPTIONAL REGULATION

这项建议主要关注蛋白质--蛋白质和蛋白质--DNA 单纯疱疹病毒转录因子VP16的相互作用 病毒病毒粒子组件，感染后负责增强 病毒即刻早期基因的表达。这样做的目的是 提案有两个方面。首先，他们的目的是理解 详述VP16中的蛋白质-蛋白质和蛋白质-DNA相互作用 参与涉及人类同源结构域蛋白Oct-1的相互作用， 另一种宿主细胞蛋白称为HCF，以及TAATGARAT顺式元件。 其次，它们旨在确定类似的机制是否 在正常的细胞转录过程中运行。后一种实验 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同源结构域，并且VP16和Oct-1结合 TAATGARAT序列中相邻的顺式亚基。这些研究 在特定目标1中描述的使用足迹和蛋白质-DNA 用于识别OCT-1和OCT-1识别的序列的交联技术 TAATGARAT顺式元件中的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，他们将使用定点突变来 确定与OCT-1相关的VP16区域 同源结构域、HCF和DNA。并测试一种假设，即一个独立的 VP16亚域负责OCT-1同源结构域的识别。 具体目标3涉及目前已知的HCF的身份 仅作为色谱组分。人绒毛膜促性腺激素将从哺乳动物中提纯 和果蝇提取物通过蛋白质亲和力和 常规的层析技术。或者，如果净化 证实困难，编码HCF活性的cDNA将通过基因克隆 酵母中的互补作用。最后，设计了特定的目标4来测试 假设类似于VP16和OCT-1的相互作用 同源结构域发生在正常的细胞转录过程中。这些研究 将基于OCT-1中涉及的VP16区域的鉴定 同源结构域关联；如果起作用的细胞转录因子 类似于VPI6存在，该同源域识别亚域可以 一直保存在它们中间。将采用两种策略来 利用这一潜在的保护。首先，聚合酶链式反应技术 用于识别细胞中类似的蛋白质编码区，以及 第二，针对VP16同源结构域识别的抗血清 亚区将用于筛选表达文库。如果这两个人 策略被证明是不成功的，这是一种使用小说的替代方法 “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.