VP16 AND HOMEODOMAIN-MEDIATED TRANSCRIPTIONAL REGULATION

VP16 和同源域介导的转录调控

• 批准号: 3308000
• 负责人: Seth Stern
• 金额: $ 20.01万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308000
• 关键词: DNA binding protein; DNA footprinting; Drosophilidae; crosslink; gene expression; genetic library; genetic regulatory element; genetic transcription; herpes simplex virus 1; host organism interaction; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis; transcription factor; transposon /insertion element; virus genetics; virus infection mechanism; yeasts

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.

该提案重点是蛋白质 - 蛋白质和蛋白质 - -DNA 病毒转录因子VP16的相互作用，单纯疱疹 病毒病毒成分，感染后负责增强 病毒直接与早期基因的表达。 目标的目标 提案是两倍。 首先，他们是针对理解的 详细说明蛋白质 - 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， 另一种宿主细胞蛋白称为HCF和Taatgarat顺元。 其次，它们旨在确定是否类似机制 在正常细胞转录期间运行。 后一个实验 将以最近的研究结果为指导，这些结果表明 VPI6可以识别Oct-1同源域，并且VP16和OCT-1绑定 taatgarat序列中的相邻顺式蛋白。 研究 在特定目标中描述的1采用足迹和蛋白质-DNA 交联技术，以识别Oct-1和Oct-1识别的序列 taatgarat顺元中的VP16。 特定描述的研究 AIM 2旨在了解VP16功能的结构基础。 除其他技术外，它们还将采用以网站为导向的诱变 确定负责与Oct-1关联的VP16区域 同源域，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。 特定目标3与HCF的身份有关，目前已知 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，cDNA的编码HCF活性将被遗传克隆 酵母中的互补。 最后，特定目标4旨在测试 假设相互作用类似于VP16和OCT-1的相互作用 正常细胞转录期间出现同源域。 这些研究 将基于对涉及Oct-1的VP16区域的识别 同源域协会；如果功能的细胞转录因子 与VPI6类似，此副域 - 识别子域可能 在其中保存了。 将采用两种策略 利用这种潜在的保护。 首先，PCR技术将是 用于鉴定细胞cDNA中的相似蛋白质编码区，并 第二，反对VP16同源域识别的Antisera提出 亚域将用于筛选cDNA表达文库。 如果这两个 策略证明是不成功的，是一种使用小说的替代方法 将采用“融合型” cDNA克隆技术来检测蛋白质 可以与同源域进行互动。