TRANS-ACTIVATED EXPRESSION OF HSV IMMEDIATE EARLY GENES

转录调控是许多生物学中的关键步骤 真核细胞中的过程，包括控制细胞生长和 组织器官的分裂、分化和发育； 对细胞外信号和环境条件的反应。这个 这个项目的长期目标是了解分子 转录激活机制。疱疹裂解性感染 单纯性病毒代表了这一目标的一个有用的模型系统，在 单纯疱疹病毒病毒粒子(称为VP16)的一个成分 有效地激活病毒的即刻早期基因。激活 VP16的结构域是迄今为止已知的最有效的结构域之一，因此一直是 被广泛用作研究这一现象的范例。 本项目期间的具体目标将采用广泛的 生物化学和分子遗传学方法探索关键基因 VP16转录激活结构域的结构特征。 将使用寡核苷酸定向突变来检测特定的 从以前的工作中出现的关于特定角色的假说 氨基酸或结构基序。确定VP16的关键功能 可能不是当前假设目标的函数，随机地- 产生的突变体将通过体内选择和 酵母菌中的筛选试验。辅助服务器和辅助服务器的物理描述 将使用差示扫描来寻找VP16的三级结构 量热法、傅里叶变换红外光谱、圆二色谱 光谱学，二维核磁共振，和 结晶学技术最后，身份识别和 经济和非典型肺炎相关病毒VP16同源物的特性研究 将追求农业重要性，以揭示结构特征 通过它们经历的自然选择事件而保存下来 病毒。 这个项目不仅将大大增加对 转录激活机制，但也将提供 探索人类和人类的抗病毒策略所需的知识 兽医使用和开发单纯疱疹病毒病媒作为疫苗 选择性基因治疗。

The regulation of transcription is a key step in many biological processes in eukaryotic cells, including control of cell growth and division, differentiation and development of tissues and organs, and response to extracellular signals and environmental conditions. The long-term objective of this project is an understanding of the molecular mechanisms of transcriptional activation. Lytic infection by herpes simplex virus represents a useful model system for this objective, in that a component of the HSV virion (termed VP16) specifically and potently activates of the viral immediate-early genes. The activation domain of VP16 is among the most potent known to date, and thus has been widely adopted as a paradigm for the study of this phenomenon. The specific aims for this project period will employ a broad range of biochemical and molecular genetic methods to explore the critical structural features of the VP16 transcriptional activation domain. Oligonucleotide-directed mutagenesis will be used to test specific hypotheses emerging from previous work regarding the roles of particular amino acids or structural motifs. To identify features critical to VP16 function that might not be targeted by current hypotheses, randomly- generated mutants will be identified through in vivo selection and screening assays in yeast. Physical descriptions of the secondary and tertiary structures of VP16 will be sought using differential scanning calorimetry, Fourier-transform infrared spectroscopy, circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance, and crystallographic techniques. Finally, identification and characterization of VP16 homologs in related viruses of economic and agricultural importance will be pursued to reveal structural features conserved through the natural selection events undergone by these viruses. This project will not only add significantly to an understanding of the mechanisms of transcriptional activation, but will also provide knowledge necessary to explore antiviral strategies for human and veterinary use and for exploitation of HSV vectors as agents for selective gene therapy.